Animal models and therapeutic molecules

ABSTRACT

The invention discloses methods for the generation of chimaeric human-non-human antibodies and chimaeric antibody chains, antibodies and antibody chains so produced, and derivatives thereof including fully humanised antibodies; compositions comprising said antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in said methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 13/740,727, filed Dec. 2, 2011, which is a Continuation-in-Part ofPCT/GB2010/051122, filed Jul. 7, 2010, which claims the benefit of U.S.Provisional Application No. 61/223,960 filed Jul. 8, 2009; U.S.Provisional Application No. 61/355,666, filed Jun. 17, 2010; GB PatentApplication No. 0911846.4, filed Jul. 8, 2009; and GB Patent ApplicationNo. 0913102.0, filed Jul. 28, 2009. U.S. Ser. No. 13/310,431 is also aContinuation-in-Part of PCT/GB2011/050019, filed Jan. 7, 2011. Theentire contents of the above-referenced applications are incorporatedherein by reference.

BACKGROUND

The present invention relates inter alia to non-human animals and cellsthat are engineered to contain exogenous DNA, such as humanimmunoglobulin gene DNA, their use in medicine and the study of disease,methods for production of non-human animals and cells, and antibodiesand antibody chains produced by such animals and derivatives thereof.

In order to get around the problems of humanizing antibodies a number ofcompanies set out to generate mice with human immune systems. Thestrategy used was to knockout the heavy and light chain loci in ES cellsand complement these genetic lesions with transgenes designed to expressthe human heavy and light chain genes. Although fully human antibodiescould be generated, these models have several major limitations:

(i) The size of the heavy and light chain loci (each several Mb) made itimpossible to introduce the entire loci into these models. As a resultthe transgenic lines recovered had a very limited repertoire ofV-regions, most of the constant regions were missing and importantdistant enhancer regions were not included in the transgenes.

(ii) The very low efficiency of generating the large insert transgeniclines and the complexity and time required to cross each of these intothe heavy and light chain knockout strains and make them homozygousagain, restricted the number of transgenic lines which could be analysedfor optimal expression.

(iii) Individual antibody affinities rarely reached those which could beobtained from intact (non-transgenic) animals.

WO2007117410 discloses chimaeric constructs for expressing chimaericantibodies.

WO2010039900 discloses knock in cells and mammals having a genomeencoding chimaeric antibodies.

The present invention provides, inter alia, a process for the generationin non-human mammals of antibodies that comprise a human Ig variableregion, and further provides non-human animal models for the generationof such antibodies.

SUMMARY OF THE INVENTION

All nucleotide co-ordinates for the mouse are those corresponding toNCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEMBL Release55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 seeWorld Wide Web (www) genome.ucsc.edu and World Wide Web (www)genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified. Humannucleotides coordinates are those corresponding to GRCh37 (e.g. UCSCversion hg 19, World Wide Web (www)genome.ucsc.edu/FAQ/FAQreleases.html), February 2009 ENSEMBL Release55.37, or are those corresponding to NCBI36, Ensemble release 54 unlessotherwise specified. Rat nucleotides are those corresponding to RGSC 3.4Dec. 2004 ENSEMBL release 55.34w, or Baylor College of Medicine HGSCv3.4 Nov. 2004 (e.g., UCSC rn4, see World Wide Web (www) genome.ucsc.eduand World Wide Web (www)genome.ucsc.edu/FAQ/FAQreleases.html) unlessotherwise specified.

In the present invention, methods are disclosed for constructing achimaeric human heavy and light chain loci in a non-human mammal, forexample a mouse. Reference to work in mice herein is by way of exampleonly, and reference to mice is taken to include reference to allnon-human mammals unless otherwise apparent from the disclosure, withmice being preferred as the non-human mammal.

In one aspect the invention relates to a non-human mammal whose genomecomprises:

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) optionally one or more human Ig light chain kappa V regions        and one or more human Ig light chain kappa J regions upstream of        the host non-human mammal kappa constant region and/or one or        more human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies, or chimaeric light or heavy chains, having anon-human mammal constant region and a human variable region.

In one aspect the invention relates to non-human mammal whose genomecomprises

-   -   (a) a plurality of human Ig light chain kappa V regions and one        or more human Ig light chain kappa J regions upstream of the        host non-human mammal kappa constant region and/or a plurality        of human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region; and    -   (b) optionally one or more human IgH V regions, one or more        human D regions and one or more human J regions upstream of the        host non-human mammal constant region;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies, or chimaeric light or heavy chains, having anon-human mammal constant region and a human variable region.

In one aspect the invention relates to non-human mammalian cell whosegenome comprises

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region and    -   (b) optionally one or more human Ig light chain kappa V regions        and one or more human Ig light chain kappa J regions upstream of        the host non-human mammal kappa constant region and/or one or        more human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region.

In one aspect the invention relates to a non-human mammalian cell whosegenome comprises

-   -   (a) a plurality of human Ig light chain kappa V regions and one        or more human Ig light chain kappa J regions upstream of the        host non-human mammal kappa constant region and/or a plurality        of human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region; and    -   (b) optionally one or more human IgH V regions, one or more        human D regions and one or more human J regions upstream of the        host non-human mammal constant region;

In a further aspect the invention relates to a method for producing anon-human cell or mammal comprising inserting into a non-human mammalcell genome, such as an ES cell genome;

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) optionally one or more human Ig light chain kappa V regions        and one or more human Ig light chain kappa J regions upstream of        the host non-human mammal kappa constant region and/or one or        more human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region; respectively, the insertion being        such that the non-human cell or mammal is able to produce a        repertoire of chimaeric antibodies having a non-human mammal        constant region and a human variable region, wherein steps (a)        and (b) can be carried out in either order and each of steps (a)        and (b) can be carried out in a stepwise manner or as a single        step. Insertion may be by homologous recombination.

In a further aspect the invention relates to a method for producing anantibody or antibody chain specific to a desired antigen the methodcomprising immunizing a transgenic non-human mammal as disclosed hereinwith the desired antigen and recovering the antibody or antibody chain.

In a further aspect the invention relates to a method for producing afully humanised antibody comprising immunizing a transgenic non-humanmammal as disclosed herein with the desired antigen, recovering theantibody or cells producing the antibody and then replacing thenon-human mammal constant region with a human constant region, forexample by protein or DNA engineering.

In a further aspect the invention relates to humanised antibodies andantibody chains produced according to the present invention, both inchimaeric (for example, mouse-human) and fully humanised form, as wellas fragments and derivatives of said antibodies and chains, and use ofsaid antibodies, chains and fragments in medicine, including diagnosis.

In a further aspect the invention relates to use of a non-human mammalas described herein as a model for the testing of drugs and vaccines.

In one aspect the invention relates to a non-human mammal whose genomecomprises:

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) optionally one or more human Ig light chain kappa V regions        and one or more human Ig light chain kappa J regions upstream of        the host non-human mammal kappa constant region and/or one or        more human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies or antibody chains having a non-human mammalconstant region and a human variable region.

In a further aspect the invention relates to a non-human mammal whosegenome comprises:

-   -   (a) a plurality of human Ig light chain kappa V regions and one        or more human Ig light chain kappa J regions upstream of the        host non-human mammal kappa constant region and/or a plurality        of human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region; and    -   (b) optionally one or more human IgH V regions, one or more        human D regions and one or more human J regions upstream of the        host non-human mammal constant;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies having a non-human mammal constant region and ahuman variable region.

Optionally the non-human mammal genome is modified to prevent expressionof fully host-species specific antibodies.

In one aspect the inserted human DNA comprises at least 50% of the humanheavy chain variable (V) genes, such as at least 60%, at least 70%, atleast 80%, at least 90%, and in one aspect all of the human V genes.

In one aspect the inserted human DNA comprises at least 50% of the humanheavy chain diversity (D) genes, such as at least 60%, at least 70%, atleast 80%, at least 90%, and in one aspect all of the human D genes.

In one aspect the inserted human DNA comprises at least 50% of the humanheavy chain joining (J) genes, such as at least 60%, at least 70%, atleast 80%, at least 90%, and in one aspect all of the human J genes.

In one aspect the inserted human DNA comprises at least 50% of the humanlight chain Variable (V) genes, such as at least 60%, at least 70%, atleast 80%, at least 90%, and in one aspect all of the human light chainV genes.

In one aspect the inserted human DNA comprises at least 50% of the humanlight chain joining (J) genes, such as at least 60%, at least 70%, atleast 80%, at least 90%, and in one aspect all of the human light chainJ genes.

The inserted human genes may be derived from the same individual ordifferent individuals, or be synthetic or represent human consensussequences.

Although the number of V D and J regions is variable between humanindividuals, in one aspect there are considered to be 51 human V genes,27 D and 6 J genes on the heavy chain, 40 human V genes and 5 J genes onthe kappa light chain and 29 human V genes and 4 J genes on the lambdalight chain (Janeway and Travers, Immunobiology, Third edition)

In one aspect the human heavy chain locus inserted into the non-humanmammal contains the full repertoire of human V, D and J regions, whichin the genome is in functional arrangement with the non-human mammalconstant regions such that functional chimaeric antibodies can beproduced between the human variable and non-human mammal constantregions. This total inserted human heavy chain genetic material isreferred to herein as the human IgH VDJ region, and comprises DNA from ahuman genome that encodes all the exons encoding human V,D and Jportions and suitably also the associated introns. Similarly, referenceto the human Ig light chain kappa V and J regions herein refers to humanDNA comprising all the exons encoding V and J regions and suitably alsothe associated introns of the human genome. Reference to the human Iglight chain lambda V and J regions herein refers to human DNA comprisingall the exons encoding V and J regions and suitably also the associatedintrons of the human genome.

Human variable regions are suitably inserted upstream of a non-humanmammal constant region, the latter comprising all of the DNA required toencode the full constant region or a sufficient portion of the constantregion to allow the formation of an effective chimaeric antibody capableof specifically recognising an antigen.

In one aspect the chimaeric antibodies or antibody chains have a part ofa host constant region sufficient to provide one or more effectorfunctions seen in antibodies occurring naturally in a host mammal, forexample that they are able interact with Fc receptors, and/or bind tocomplement.

Reference to a chimaeric antibody or antibody chain having a host nonmammal constant region herein therefore is not limited to the completeconstant region but also includes chimaeric antibodies or chains whichhave all of the host constant region, or a part thereof sufficient toprovide one or more effector functions. This also applies to non-humanmammals and cells and methods of the invention in which human variableregion DNA may be inserted into the host genome such that it forms achimaeric antibody chain with all or part of a host constant region. Inone aspect the whole of a host constant region is operably linked tohuman variable region DNA.

The host non-human mammal constant region herein is preferably theendogenous host wild-type constant region located at the wild typelocus, as appropriate for the heavy or light chain. For example, thehuman heavy chain DNA is suitably inserted on mouse chromosome 12,suitably adjacent the mouse heavy chain constant region.

In one aspect the insertion of the human DNA, such as the human VDJregion is targeted to the region between the J4 exon and the Cμ locus inthe mouse genome IgH locus, and in one aspect is inserted betweenco-ordinates 114,667,090 and 114,665,190, or at co-ordinate 114,667,091,after 114,667,090. In one aspect the insertion of the human DNA, such asthe human light chain kappa VJ is targeted into mouse chromosome 6between co-ordinates 70,673,899 and 70,675,515, suitably at position70,674,734, or an equivalent position in the lambda mouse locus onchromosome 16.

In one aspect the host non-human mammal constant region for forming thechimaeric antibody may be at a different (non endogenous) chromosomallocus. In this case the inserted human DNA, such as the human variableVDJ or VJ region(s) may then be inserted into the non-human genome at asite which is distinct from that of the naturally occurring heavy orlight constant region. The native constant region may be inserted intothe genome, or duplicated within the genome, at a different chromosomallocus to the native position, such that it is in a functionalarrangement with the human variable region such that chimaericantibodies of the invention can still be produced.

In one aspect the human DNA is inserted at the endogenous host wild-typeconstant region located at the wild type locus between the host constantregion and the host VDJ region.

Reference to location of the variable region upstream of the non-humanmammal constant region means that there is a suitable relative locationof the two antibody portions, variable and constant, to allow thevariable and constant regions to form a chimaeric antibody or antibodychain in vivo in the mammal. Thus, the inserted human DNA and hostconstant region are in functional arrangement with one another forantibody or antibody chain production.

In one aspect the inserted human DNA is capable of being expressed withdifferent host constant regions through isotype switching. In one aspectisotype switching does not require or involve trans switching. Insertionof the human variable region DNA on the same chromosome as the relevanthost constant region means that there is no need for trans-switching toproduce isotype switching.

As explained above, the transgenic loci used for the prior art modelswere of human origin, thus even in those cases when the transgenes wereable to complement the mouse locus so that the mice produced B-cellsproducing fully human antibodies, individual antibody affinities rarelyreached those which could be obtained from intact (non-transgenic)animals. The principal reason for this (in addition to repertoire andexpression levels described above) is the fact that the control elementsof the locus are human. Thus, the signalling components, for instance toactivate hyper-mutation and selection of high affinity antibodies arecompromised.

In contrast, in the present invention, host non-human mammal constantregions are maintained and it is preferred that at least one non-humanmammal enhancer or other control sequence, such as a switch region, ismaintained in functional arrangement with the non-human mammal constantregion, such that the effect of the enhancer or other control sequence,as seen in the host mammal, is exerted in whole or in part in thetransgenic animal.

This approach above is designed to allow the full diversity of the humanlocus to be sampled, to allow the same high expression levels that wouldbe achieved by non-human mammal control sequences such as enhancers, andis such that signalling in the B-cell, for example isotype switchingusing switch recombination sites, would still use non-human mammalsequences.

A mammal having such a genome would produce chimaeric antibodies withhuman variable and non-human mammal constant regions, but these could bereadily humanized, for example in a cloning step. Moreover the in vivoefficacy of these chimaeric antibodies could be assessed in these sameanimals.

In one aspect the inserted human IgH VDJ region comprises, in germlineconfiguration, all of the V, D and J regions and intervening sequencesfrom a human.

In one aspect 800-1000 kb of the human IgH VDJ region is inserted intothe non-human mammal IgH locus, and in one aspect a 940, 950 or 960 kbfragment is inserted. Suitably this includes bases 105,400,051 to106,368,585 from human chromosome 14.

In one aspect the inserted IgH human fragment consists of bases105,400,051 to 106,368,585 from chromosome 14. In one aspect theinserted human heavy chain DNA, such as DNA consisting of bases105,400,051 to 106,368,585 from chromosome 14, is inserted into mousechromosome 12 between the end of the mouse J4 region and the Eμ region,suitably between co-ordinates 114,667,090 and 114,665,190, or atco-ordinate 114,667,091, after 114,667,090. In one aspect the insertionis between co-ordinates 114,667,089 and 114,667,090 (co-ordinates referto NCBI m37, for the mouse C57BL/6J strain), or at equivalent positionin another non-human mammal genome.

In one aspect the inserted human kappa VJ region comprises, in germlineconfiguration, all of the V and J regions and intervening sequences froma human. Suitably this includes bases 88,940,356 to 89,857,000 fromhuman chromosome 2, suitably approximately 917 kb. In a further aspectthe light chain VJ insert may comprise only the proximal clusters of Vsegments and J segments. Such an insert would be of approximately 473kb. In one aspect the human light chain kappa DNA, such as the human IgKfragment of bases 88,940,356 to 89,857,000 from human chromosome 2, issuitably inserted into mouse chromosome 6 between co-ordinates70,673,899 and 70,675,515, suitably at position 70,674,734. Theseco-ordinates refer to NCBI36 for the human genome, ENSEMBL Release 54and NCBIM37 for the mouse genome, relating to mouse strain C57BL/6J.

In one aspect the human lambda VJ region comprises, in germlineconfiguration, all of the V and J regions and intervening sequences froma human.

Suitably this includes analogous bases to those selected for the kappafragment, from human chromosome 2.

A cell or non-human mammal of the invention, in one embodiment,comprises an insertion of human heavy chain variable region DNA betweenco-ordinates 114, 666, 183 and 114, 666, 725, such as between 114 666283 and 114 666 625, optionally between co-ordinates 114,666,335 and114,666,536, optionally between 114,666,385 and 114,666,486, or between114,666,425 and 114,666,446, or between 114,666,435 and 114,666,436 ofmouse chromosome 12 with reference to NCBIM37 for the mouse genome,relating to mouse strain C57BL/6J or an equivalent position of mousechromosome 12 from a different mouse strain or an equivalent position inthe genome of another non-human vertebrate, e.g., a rat. The insertionbetween co-ordinates 114,666,435 and 114,666,436 relating to mousestrain C57BL/6J is equivalent to an insertion between co-ordinates1207826 and 1207827 on chromosome 12 with reference to the 129/SvJgenomic sequence of the GenBank® access number NT114985.2. An insertionmay be made at equivalent position in another genome, such as anothermouse genome. In an example of this embodiment, the cell or mammal ofthe invention comprises a human IgH VDJ region which comprises orconsists of nucleotides 106,328,851-107,268,544, such as nucleotides106,328,901-107,268,494, such as nucleotides 106,328,941-107,268,454,such as nucleotides 106,328,951-107,268,444 of human Chromosome 14, withreference to the GRCH37/hg19 sequence database, or insertion ofequivalent nucleotides relating to chromosome 14 from a different humansequence or database. The human insertion may be made between theregions indicated above.

A cell or mammal of the invention, in one embodiment, comprises aninsertion of the human kappa VJ region, suitably comprising orconsisting of, in germline configuration, all of the V and J regions andintervening sequences from a human, the insertion of the human DNA beingmade between co-ordinates 70,673,918-70,675,517, such as betweenco-ordinates 70, 674,418 and 70 675, 017, such as between co-ordinates70,674, 655-70,674,856, such as between co-ordinates 70,674,705-70,674,906, such as between co-ordinates 70,674, 745-70,674,766,such as between co-ordinates 70,674,755 and 70,674,756 of mousechromosome 6, numbering with reference to NCBIM37 for the mouse genome,relating to mouse strain C57BL/6J, or an insertion at an equivalentposition in another genome, such as another mouse genome. In an exampleof this embodiment, a cell or mammal of the invention comprises aninsertion of nucleotides 89,159,079-89,630,437 and/or89,941,714-90,266,976 of human chromosome 2 with reference to theGRCH37/hg19 sequence database (or equivalent nucleotides relating tochromosome 2 from a different human sequence or database), such as aninsertion of these 2 discrete fragments without the interveningsequence, or an insertion of the complete 89,159,079-90,266,976 region.

The insertion may comprise, or consist, of:

-   -   (i) nucleotides 89,158,979-89,630,537, such as        89,159,029-89,630,487, such as 89,159,069-89,630,447, such as        89,159,079-89,630,437, optionally in addition to fragment (ii)        below    -   (ii) nucleotides 89,941,614-90,267,076, such as        89,941,664-90,267,026, such as 89, 941,704-90,266,986, such as        89,941,714-90,266,976; optionally in addition to fragment (i)    -   (iii) nucleotides 89,158,979-90,267,076, such as nucleotides        89,159,079-90,266,976.

The human insertion may be made between the regions indicated above.

In an embodiment, a cell or mammal of the invention comprises aninsertion of a human lambda region which comprises at least one human JAregion (eg, a germline region) and at least one human Cλ region (eg, agermline region), optionally C_(λ)6 and/or C_(λ)7. For example, the cellor mammal comprises a plurality of human JA regions, optionally two ormore of J_(λ)1, J_(λ)2, J_(λ)6 and J_(λ)7, optionally all of J_(λ)1,J_(λ)2, J_(λ)6 and J_(λ)7. In an example, the cell or mammal comprisesat least one human J_(λ)-C_(λ) cluster, optionally at leastJ_(λ)7-C_(λ)7.

In one aspect the human JC cluster is inserted 3′ of the last endogenousJ lambda or is inserted 3′ of the last endogenous J kappa region,suitably immediately 3′ of these sequences, or substantially immediately3′ of these sequences.

In one aspect the insertion into the mouse lambda locus is madedownstream of the endogenous C1 gene segment, for example where there isa 3′ J1C1 cluster, suitably immediately 3′ of the C1 segment, orsubstantially immediately 3′ of the segment.

In one aspect (e.g. cell or non-human mammal) a human JC cluster isinserted into a kappa locus and any resulting cell or animal isheterozygous at that locus, such that the cell has one chromosome withhuman lambda DNA inserted into the kappa locus, and another chromosomewith human kappa DNA at the endogenous kappa locus.

In an embodiment, a cell or mammal of the invention comprises a human EAenhancer.

A cell or mammal may of the invention comprise an inserted human lambdaVJ region, suitably comprising or consisting of, in germlineconfiguration, all of the V and J regions and intervening sequences froma human, the inserted region comprises or consisting of nucleotides22,375,509-23,327,984, such as nucleotides 22,375,559-23,327,934, suchas nucleotides 22,375,599-23,327,894, such as nucleotides22,375,609-23,327,884 from human Chromosome 22, with reference to theGRCH37/hg19 sequence database, or equivalent DNA from another humansequence or database. The insertion into the mouse genome may be madebetween co-ordinates 19,027,763 and 19,061,845, such as betweenco-ordinates 19, 037, 763 and 19, 051, 845, such as between co-ordinates19,047,451 and 19,047,652, such as between co-ordinates 19,047,491 and19,047,602, such as between co-ordinates 19,047,541 and 19,047,562, suchas between co-ordinates 19,047,551 and 19,047,552 of mouse Chromosome 16(with reference to NCBIM37 for the mouse genome, relating to mousestrain C57BL/6J, equivalent to co-ordinates 1,293,646-1,293,647 of the129 SvJ genomic sequence in the sequence file of NT 039630.4), or may bean insertion at an equivalent position in other genome, such as anothermouse genome. The insertion of the human lambda nucleic acid into themouse genome may alternatively be made between co-ordinates 70,673,918and 70,675,517, such as between co-ordinates 70, 674,418 and 70 675,017, such as between co-ordinates 70,674,655 and 70,674,856, such asbetween co-ordinates 70,674,705 and 70,674,806, such as betweenco-ordinates 70,674,745 and 70,674,766, such as between co-ordinates70,674,755 and 70,674,756 of mouse Chromosome 6 (with reference toNCBIM37 for the mouse genome, relating to mouse strain C57BL/6J) orequivalent in another genome. The human insertion may be made betweenthe regions indicated above.

All specific human fragments described above may vary in length, and mayfor example be longer or shorter than defined as above, such as 500bases, 1 KB, 2K, 3K, 4K, 5 KB, 10 KB, 20 KB, 30 KB, 40 KB or 50 KB ormore, which suitably comprise all or part of the human V(D)J region,whilst preferably retaining the requirement for the final insert tocomprise human genetic material encoding the complete heavy chain regionand light chain region, as appropriate, as described above.

In one aspect the 5′ end of the human insert described above isincreased in length. Where the insert is generated in a stepwise fashionthen the increase in length is generally in respect of the upstream (5′)clone.

In one aspect the 3′ end of the last inserted human gene, generally thelast human J gene to be inserted is less than 2 kb, preferably less than1 KB from the human-mouse join region.

In one aspect the non-human mammal comprises some or all of the humanlight chain kappa VJ region as disclosed herein but not the human lightchain lambda VJ region.

In one aspect the cell or non-human mammal comprises a fully humanlambda locus (lambda VJC regions from a human), a chimaeric kappa locus(human kappa VJ regions operatively linked to a host kappa constantregion) and a chimaeric heavy chain locus, having a human VDJ regionoperatively linked to a host heavy chain constant region.

In a further aspect the genome comprises an insertion of V, D (heavychain only) and J genes as described herein at the heavy chain locus andone light chain locus, or at the heavy chain locus and both light chainloci. Preferably the genome is homozygous at one, or both, or all threeloci.

In another aspect the genome may be heterozygous at one or more of theloci, such as heterozygous for DNA encoding a chimaeric antibody chainand native (host cell) antibody chain. In one aspect the genome may beheterozygous for DNA capable of encoding 2 different antibody chains ofthe invention, for example, comprising 2 different chimaeric heavychains or 2 different chimaeric light chains.

In one aspect the invention relates to a non-human mammal or cell, andmethods for producing said mammal or cell, as described herein, whereinthe inserted human DNA, such as the human IgH VDJ region and/or lightchain V, J regions are found on only one allele and not both alleles inthe mammal or cell. In this aspect a mammal or cell has the potential toexpress both an endogenous host antibody heavy or light chain and achimaeric heavy or light chain.

In a further aspect of the invention the human VDJ region, or lightchain VJ region, is not used in its entirety, but parts of theequivalent human VDJ or VJ region, such as the exons, from other speciesmay be used, such as one or more V, D, or J exons from other species, orregulatory sequences from other species. In one aspect the sequencesused in place of the human sequences are not human or mouse. In oneaspect the sequences used may be from rodent, or, primate such as chimp.For example, 1, 2, 3, 4, or more, or all of the J regions from a primateother than a human may be used to replace, one, 2, 3, 4, or more or allof the human J exons in the VDJ/VJ region of the cells and animals ofthe invention.

In a further aspect the inserted human DNA, such as the human IgH VDJregion, and/or light chain VJ regions, may be inserted such that theyare operably linked in the genome with a mu constant region from anon-human, non-mouse species, such as a rodent or primate sequence, suchas a rat sequence.

Other non-human, non-mouse species from which DNA elements may be usedin the present invention include rabbits, lamas, dromedary, alpacas,camels and sharks.

In one aspect the inserted human DNA, such as the human VDJ or VJregion, is not operably linked to the endogenous host mu sequence butrather to a non-host mu sequence.

Operable linkage suitably allows production of an antibody heavy orlight chain comprising the human variable region.

In one aspect the inserted human DNA, such as the human IgH VDJ region(and/or light chain VJ regions) may be inserted into the host chromosometogether with mu constant region nucleic acid which is not host muconstant region nucleic acid, and preferably is a mu constant regionfrom a non-mouse, non-human species. Suitably the inserted human DNA,such as the human VDJ region (and/or light chain VJ regions) is operablylinked to a non-human, non-mouse mu, and is able to form a chimaericantibody heavy or light chain. In another aspect a non-mouse, non-humanmu may be inserted into the host chromosome on a separate geneticelement to that of the human variable region, or at a different locationin the genome, suitably operably linked to the variable region such thata chimaeric antibody heavy or light can be formed.

In an additional aspect the invention relates to a non-human mammal or acell whose genome comprises a plurality of human IgH V regions, one ormore human D regions and one or more human J regions upstream of a hostnon-human mammal light chain constant region, arranged such that thecell or mammal is able to express a chimaeric antibody chain. Theinvention also relates to a non-human mammal or a cell whose genomeadditionally or alternatively comprises a plurality of human Ig lightchain V regions, and one or more human J regions upstream of a hostnon-human mammal heavy chain constant region, such that the cell ormammal is able to express a chimaeric antibody chain. The cell or mammalmay be able to express an antibody having both heavy and light chains,including at least one chimaeric antibody chain, as disclosed above.

The inserted human heavy chain variable regions may be any of thosedescribed herein, and may be inserted at the positions described abovefor insertion 5′ of the lambda and kappa constant regions. Likewise theinserted human light chain variable regions may be those describedabove, and may be inserted at the positions described above forinsertion 5′ of the heavy chain constant region.

For example, the genome or the cell or non-human mammal of the inventionmay encode an antibody comprising an antibody chain having a human heavychain variable region upstream of a mouse light chain constant region,or an antibody chain having a human light chain variable region upstreamof a mouse heavy chain constant region, in combination with one of:

-   -   a fully human antibody light chain;    -   a fully human antibody heavy chain;    -   a non-human vertebrate (e.g., mouse or rat) antibody light        chain;    -   a non-human vertebrate (e.g., mouse or rat) antibody heavy        chain;    -   a chimaeric non-human vertebrate (e.g., mouse or rat)—human        antibody chain;    -   an antibody chain having a human heavy chain variable region        upstream of a non-human vertebrate (e.g., mouse or rat) light        chain constant region;    -   an antibody chain having a human light chain variable region        upstream of a non-human vertebrate (e.g., mouse or rat) heavy        chain constant region.

The invention also relates to a transgene encoding a plurality of humanIgH V regions, one or more human D regions and one or more human Jregions upstream of a host non-human mammal light chain constant region,optionally comprised within a vector.

The invention also relates to a transgene encoding a plurality of humanIg light chain V regions, and one or more human light chain J regionsupstream of a host non-human mammal heavy chain constant region,optionally comprised within a vector.

In one aspect the invention relates to a cell, or non-human mammal, thegenome of which comprises: one or more human Ig light chain kappa Vregions and one or more human Ig light chain kappa J regions upstream ofall or part of the human kappa constant region.

In another aspect the invention relates to a cell, or non-human mammal,the genome of which comprises: one or more human Ig light chain lambda Vregions and one or more human Ig light chain lambda J regions upstreamof all or part of the human lambda constant region.

Suitably the light chain VJ and C regions are able to form antibodychains in vivo capable of specifically reacting with an antigen.

In one aspect of the invention there is no non-human coding sequence inthe inserted light chain region.

In such aspects a human kappa and/or lambda region is inserted into thegenome, in combination with insertion of the heavy chain VDJ region orpart thereof, upstream of the host heavy chain constant region asdisclosed herein.

The cell or non-human mammal of the invention may comprise:

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) one or more human Ig light chain kappa V regions and one or        more human Ig light chain kappa J regions upstream of all or        part of the non-human kappa constant region, wherein the        non-human mammal is able to produce a repertoire of antibodies        having an antibody chain comprising non-human mammal constant        region and a human variable region.

The cell or non-human mammal of the invention may comprise

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   one or more human Ig light chain lambda V regions and one or        more human Ig light chain lambda J regions upstream of the host        non-human mammal lambda constant region; wherein the non-human        mammal is able to produce a repertoire of antibodies having an        antibody chain comprising a non-human mammal constant region and        a human variable region.

Suitably the insertion of the human VJC light chain DNA, or part thereofas disclosed above, is made at the equivalent mouse locus. In one aspectthe human light chain kappa VJC DNA, or part thereof, is insertedimmediately upstream or downstream of the mouse kappa VJC region. In oneaspect, the human light chain lambda VJC region or part thereof isinserted immediately upstream or downstream of the mouse lambda VJCregion. In one aspect only the human kappa VJC locus is inserted and notthe human lambda VJC locus. In one aspect only the human lambda VJClocus is inserted and not the human kappa VJC locus. Insertions may bemade using the techniques disclosed herein, and suitably do not removethe host sequences from the genome. In one aspect the non-human mammalhost VJC sequences may be inactivated in some way, by mutation, orinversion, or by insertion of the human variable region DNA, or by anyother means. In one aspect the cell or non-human mammal of the inventionmay comprise an insertion of the complete VJC human region.

The human kappa variable region DNA might be inserted into the genome infunctional arrangement with a lambda constant region, for exampleinserted upstream of a lambda constant region. Alternatively humanlambda region variable DNA might be inserted in functional arrangementwith a kappa constant region, for example inserted upstream of a kappaconstant region.

In one aspect one or more non-human mammal control sequences such as theenhancer sequence(s) is maintained upstream of the nonhuman mammal Muconstant region, suitably in its native position with respect to thedistance from the constant region.

In one aspect one or more non-human mammal control sequences such as anenhancer sequence(s) are maintained downstream of the nonhuman mammal Muconstant region, suitably in its native position with respect to thedistance from the constant region.

In one aspect a non-human mammal switch sequence, suitably theendogenous switch sequence, is maintained upstream of the non-humanmammal Mu constant region, suitably in its native position with respectto distance from the constant region.

In such location the host enhancer or switch sequences are operative invivo with the host constant region sequence(s).

In one aspect a switch sequence is neither human, nor native in thenon-human mammal, for example in one aspect a non-human mammal switchsequence is not a mouse or human switch sequence. The switch sequencemay be, for example, a rodent or primate sequence, or a syntheticsequence. In particular the switch sequence may be a rat sequence wherethe non-human mammal is a mouse. By way of example, a mouse or humanconstant mu sequence may be placed under the control of a switchsequence from a rat, or chimp, or other switch sequence, suitablycapable of allowing isotype switching to occur in vivo.

In one aspect the switch sequence of the invention is a switch sequencecomprising 3, 4, 5, 6 or more (up to 82) contiguous repeats of therepeat sequence GGGCT (SEQ ID no 46-50), such as a rat switch sequence.By “rat switch” herein it is meant that the switch is a wild-type switchcorresponding to a switch from a rat genome or derived from such aswitch.

In one aspect the switch sequence of the invention is a rat switchsequence comprising the following repeats: GAGCT (296 repeats; SEQ ID No18), GGGGT (50 repeats; SEQ ID No 19), and GGGCT (83 repeats; SEQ ID No20).

In one example the rat switch sequence comprises or consists of thesequence of SEQ ID no 1.

In these embodiments, and where the non-human mammal is a mouse or thecell is a mouse cell, the switch is optionally a rat switch as describedherein.

Alternatively, the switch sequence present in cells or mammal of theinvention is a mouse switch, eg, is from a mouse such as a mouse 129strain or mouse C57 strain, or from a strain derived therefrom,optionally comprising or consisting of the sequence of SEQ ID no 4 or 5.By “mouse switch” herein it is meant that the switch is a wild-typeswitch corresponding to a switch from a mouse genome or derived fromsuch a switch. In this embodiment, and where the non-human mammal is amouse or the cell is a mouse cell, the mouse switch sequence isoptionally the endogenous switch or is a mouse switch from another mousestrain.

The cell or mammal of the invention may therefore comprise a human ornon-human mammal switch sequence and a human or non-human mammalenhancer region or regions. They may be upstream of a human or non-humanmammal constant region. Preferably the control sequences are able todirect expression or otherwise control the production of antibodiescomprising a constant region with which they are associated. Onecombination envisaged is a rat switch with mouse enhancer sequences andmouse constant regions in a mouse cell.

In one aspect the invention relates to a cell, preferably a non-humancell, or non-human mammal comprising an immunoglobulin heavy chain orlight chain locus having DNA from 3 or more species. For example, thecell or animal may comprise host cell constant region DNA, one or morehuman V, D or J coding sequences and one or more non-human, non-host DNAregions that are able to control a region of the immunoglobulin locus,such as a switch sequence, promoter or enhancer which are able tocontrol expression or isotype switching in vivo of the Ig DNA. In oneaspect the cell or animal is a mouse and comprises additionally humanDNA from the human Ig locus and additionally a non-mouse DNA sequence,such as a rat DNA sequence, capable of regulation of the mouse or humanDNA.

In another aspect the invention relates to a cell, preferably non-humancell, or non-human mammal comprising an immunoglobulin heavy chain orlight chain locus having DNA from 2 or more different human genomes. Forexample, it could comprise heavy chain V(D)J sequences from more thanone human genome within a heavy or light chain, or heavy chain VDJ DNAfrom one genome and light chain VJ sequences from a different genome.

In one aspect the invention relates to a DNA fragment or cell ornon-human mammal comprising an immunoglobulin heavy chain or light chainlocus, or part thereof, having DNA from 2 or more species, where onespecies contributes a non-coding region such as a regulatory region, andthe other species coding regions such as V, D, J or constant regions.

In one aspect the human promoter and/or other control elements that areassociated with the different human V, D or J regions are maintainedafter insertion of the human VDJ into the mouse genome.

In a further aspect one or more of the promoter elements, or othercontrol elements, of the human regions, such as the human V regions, areoptimised to interact with the transcriptional machinery of a non-humanmammal.

Suitably a human coding sequence may be placed under the control of anappropriate non-human mammal promoter, which allows the human DNA to betranscribed efficiently in the appropriate non-human animal cell. In oneaspect the human region is a human V region coding sequence, and a humanV region is placed under the control of a non-human mammal promoter.

The functional replacement of human promoter or other control regions bynon-human mammal promoter or control regions may be carried out by useof recombineering, or other recombinant DNA technologies, to insert apart of the human Ig region (such as a human V region) into a vector(such as a BAC) containing a non-human Ig region. Therecombineering/recombinant technique suitably replaces a portion of thenon-human (e.g. mouse) DNA with the human Ig region, and thus places thehuman Ig region under control of the non-human mammal promoter or othercontrol region. Suitably the human coding region for a human V regionreplaces a mouse V region coding sequence. Suitably the human codingregion for a human D region replaces a mouse D region coding sequence.Suitably the human coding region for a human J region replaces a mouse Jregion coding sequence. In this way human V, D or J regions may beplaced under the control of a non-human mammal promoter, such as a mousepromoter.

In one aspect the only human DNA inserted into the non-human mammaliancell or animal are V, D or J coding regions, and these are placed undercontrol of the host regulatory sequences or other (non-human, non-host)sequences, In one aspect reference to human coding regions includes bothhuman introns and exons, or in another aspect simply exons and nointrons, which may be in the form of cDNA.

It is also possible to use recombineering, or other recombinant DNAtechnologies, to insert a non-human-mammal (e.g. mouse) promoter orother control region, such as a promoter for a V region, into a BACcontaining a human Ig region. A recombineering step then places aportion of human DNA under control of the mouse promoter or othercontrol region.

The approaches described herein may also be used to insert some or allof the V, D and J regions from the human heavy chain upstream of a lightchain constant region, rather than upstream of the heavy chain constantregion. Likewise some or all of the human light chain V and J regionsmay be inserted upstream of the heavy chain constant region. Insertionmay be at the endogenous constant region locus, for example between theendogenous constant and J region, and may be of some, or all, of the V,D or J genes alone, excluding promoter or enhancer sequences, or may beof some, or all, of the V, D or J genes with one or more or allrespective promoter or enhancer sequences. In one aspect the fullrepertoire of V, D or J fragments in germline orientation may beinserted upstream and in functional arrangement with a host constantregion.

Thus the present invention allows V and/or D and/or J regions from ahuman, or any species, to be inserted into a chromosome of a cell from adifferent species that comprises a constant region, allowing a chimaericantibody chain to be expressed.

In one aspect the invention requires only that some human variableregion DNA is inserted into the genome of a non-human mammal in operablearrangement with some, or all, of the human heavy chain constant regionat the region of the endogenous heavy chain constant region locus suchthat an antibody chain can be produced. In this aspect of the inventionand where human light chain DNA is additionally inserted, the lightchain DNA insertion can be in the form of a completely human construct,having both human variable DNA and human constant region DNA, or havehuman variable region DNA and constant region DNA from a non-human,non-host species. Other variations are also possible, such as insertionof both of the light chain human variable region and host genomeconstant region. In addition the insertion of said light chaintransgenes need not be at the equivalent endogenous locus, but may beanywhere in the genome. In such a scenario the cell or mammal mayproduce chimaeric heavy chains (comprising human variable region DNA andmouse constant region DNA) and light chains comprising human variableand human constant region DNA. Thus in one aspect of the invention thelambda and or kappa human variable region DNA can be inserted upstreamof the endogenous locus, or downstream, or indeed on a differentchromosome to the endogenous locus, and inserted with or withoutconstant region DNA.

As well insertion of human light chain DNA upstream of the hostnon-human mammal constant region, a further aspect of the inventionrelates to insertion of one or both light chain human variable regionsdownstream of the equivalent endogenous locus constant region, orelsewhere in the genome.

Generally, insertion of human variable region DNA at or close to theequivalent endogenous locus in the recipient genome is preferred, forexample within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb of the boundary(upstream or downstream) of a host immunoglobulin locus.

Thus in one aspect the invention can relate to a cell or non-humanmammal whose genome comprises:

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) one or more human Ig light chain kappa V regions and one or        more human Ig light chain kappa J regions, and/or, one or more        human Ig light chain lambda V regions and one or more human Ig        light chain lambda J regions;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies, or chimaeric light or heavy chains, having anon-human mammal constant region and a human variable region.

In one particular aspect the genome of the cell or non-human mammalcomprises:

-   -   a plurality of human IgH V regions, one or more human D regions        and one or more human J regions upstream of the host non-human        mammal constant region;    -   one or more human Ig light chain kappa V regions and one or more        human Ig light chain kappa J regions upstream of the host        non-human mammal kappa constant region, and    -   one or more human Ig light chain lambda V regions and one or        more human Ig light chain lambda J regions downstream of the        host non-human mammal lambda constant region,    -   optionally in which the human lambda variable region may be        inserted upstream or downstream of the endogenous host lambda        locus in operable linkage with a human lambda constant region,        such that the non-human mammal or cell can produce fully human        antibody light chains and chimaeric heavy chains.

In a further, different, aspect of the invention, the use of the methodsof the invention allows a locus to be built up in a stepwise manner bysequential insertions, and thus allows for the insertion of humanvariable DNA together with human or non-human constant region DNA at anysuitable location in the genome of a non-human host cell. For example,methods of the invention can be used to insert human immunoglobulinvariable region DNA together with constant region DNA from the hostgenome anywhere in the genome of a non-human host cell, allowing achimaeric antibody chain to be produced from a site other than theendogenous heavy region. Any human heavy chain or light chain DNAconstruct contemplated above can be inserted into any desired positioninto the genome of a non-human host cell using the techniques describedherein. The present invention thus also relates to cells and mammalshaving genomes comprising such insertions.

The invention also relates to a vector, such as a BAC, comprising ahuman V, D or J region in a functional arrangement with a non-humanmammal promoter, or other control sequence, such that the expression ofthe human V, D or J region is under the control of the non-human mammalpromoter in a cell of the non-human mammal, such as an ES cell, inparticular once inserted into the genome of that cell.

The invention also relates to cells and non-human mammals containingsaid cells, which cells or mammals have a human V, D or J region in afunctional arrangement with a non-human mammal promoter, or othercontrol sequence, such that the expression of the human V, D or J regionis under the control of the non-human mammal promoter in the cells ormammal.

Generally, one aspect of the invention thus relates to a non-humanmammal host cell capable of expression of a human V, D or J codingsequence under the control of a host promoter or control region, theexpression capable of producing a humanised antibody having a humanvariable domain and non-human mammal constant region.

In one aspect the invention relates to a cell, such as a non mammaliancell, such as an ES cell, the genome of which comprises

-   -   (a) a plurality of human IgH V regions, one or more human D        regions and one or more human J regions upstream of the host        non-human mammal constant region; and    -   (b) optionally one or more human Ig light chain kappa V regions        and one or more human Ig light chain kappa J regions upstream of        the host non-human mammal kappa constant region and/or one or        more human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region;

In another aspect the invention relates to a cell, such as a non-humanmammal cells, such as ES cells whose genome comprises

-   -   (a) a plurality of human Ig light chain kappa V regions and one        or more human Ig light chain kappa J regions upstream of the        host non-human mammal kappa constant region and/or a plurality        of human Ig light chain lambda V regions and one or more human        Ig light chain lambda J regions upstream of the host non-human        mammal lambda constant region; and    -   (b) optionally one or more human IgH V regions, one or more        human D regions and one or more human J regions upstream of the        host non-human mammal constant region

In one aspect the cell is an ES cell is capable of developing into anon-human mammal able to produce a repertoire of antibodies which arechimaeric, said chimaeric antibodies having a non-human mammal constantregion and a human variable region. Optionally the genome of the cell ismodified to prevent expression of fully host-species specificantibodies.

In one aspect the cell is an induced pluripotent stem cell (iPS cell).

In one aspect cells are isolated non-human mammalian cells.

In one aspect a cell as disclosed herein is preferably a non-humanmammalian cell.

In one aspect the cell is a cell from a mouse strain selected fromC57BL/6, M129 such as 129/SV, BALB/c, and any hybrid of C57BL/6, M129such as 129/SV, or BALB/c.

The invention also relates to a cell line which is grown from orotherwise derived from cells as described herein, including animmortalised cell line. The cell line may comprise inserted human V, Dor J genes as described herein, either in germline configuration orafter rearrangement following in vivo maturation. The cell may beimmortalised by fusion (eg, electrofusion or using PEG according tostandard procedures.) to a tumour cell (eg, P3X63-Ag8.653 (obtainablefrom LGC Standards; CRL-1580), SP2/0-Ag14 (obtainable from ECACC), NSIor NS0), to provide an antibody producing cell and cell line, or be madeby direct cellular immortalisation.

The present invention also relates to vectors for use in the invention.In one aspect such vectors are BACs (bacterial artificial chromosomes).It will be appreciated that other cloning vectors may be used in theinvention, and therefore reference to BACs herein may be taken to refergenerally to any suitable vector.

In one aspect BACs used for generation of human DNA to be inserted, suchas the VDJ or VJ regions are trimmed so that in the final human VDJ orVJ region or part thereof in the non-human mammal, no sequence isduplicated or lost when compared to the original human genomic sequence.

In one aspect the invention relates to a vector comprising an insert,preferably comprising a region of human DNA from some of the human VDJor VJ locus, flanked by DNA which is not from that locus. The flankingDNA may comprise one or more selectable markers or one or more sitespecific recombination sites. In one aspect the vector comprises 2 ormore, such as 3, heterospecific and incompatible site specificrecombination sites. In one aspect the site specific recombination sitesmay be loxP sites, or variants thereof, or FRT sites or variantsthereof. In one aspect the vector comprises one or more transposon ITR(inverted terminal repeat) sequences.

In one aspect the non-human animals of the invention suitably do notproduce any fully humanised antibodies. In one aspect this is becausethere is no DNA inserted from the human constant region. Alternativelythere is no human constant region DNA in the genome capable of formingan antibody in conjunction with the inserted human variable region DNAcomponent, for example due to mutation within any human constant regionDNA or distance from any constant region human DNA and human variableregion DNA.

In one aspect human light chain constant region DNA may be included inthe cell genome, such that a fully human lambda or kappa human antibodychain might be generated, but this would only be able to form anantibody with a chimaeric heavy chain, and not produce a fully humanantibody having human variable and constant regions.

In one aspect the non-human mammal genome is modified to preventexpression of fully host-species specific antibodies. Fully host speciesspecific antibodies are antibodies that have both variable and constantregions from the host organism. In this context the term ‘specific’ isnot intended to relate to the binding of the antibodies produced by thecells or animals of the invention but rather to the origin of the DNAwhich encodes those antibodies.

In one aspect the non-human mammal genome is modified to preventexpression of the native (fully host species specific) antibodies in themammal by inactivation of all or a part of the host non-human mammal Igloci. In this context, inactivation or prevention of endogenous antibodyor gene segment usage (using any inactivation technique describedherein) is, for example, substantially complete inactivation orprevention (substantially 100%, ie, essentially none (eg, less than 10,5, 4, 3, 2, 1 or 0.5%) of the endogenous antibody chain (eg, noendogenous heavy chains) is expressed). This can be determined, forexample, at the antibody chain (protein) level by assessing the antibodyrepertoire produced by the non-human vertebrate, mammal or at thenucleotide level by assessing mRNA transcripts of antibody chain loci,eg, using RACE. In an embodiment, inactivation is more than 50% (ie, 50%or less of the antibodies or transcripts are of an endogenous antibodychain), 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. For example,in an embodiment, endogenous heavy chain expression is substantiallyinactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99%of the heavy chain repertoire of the vertebrate (mammal) is provided byendogenous heavy chains. For example, endogenous heavy chain expressionis substantially inactivated such that substantially none of the heavychain repertoire of the vertebrate (mammal) is provided by endogenousheavy chains. For example, in an embodiment, endogenous heavy chainexpression is substantially inactivated such that no more than 85%, 90%,95%, 96%, 97%, 98% or 99% of the kappa chain repertoire of thevertebrate (mammal) is provided by endogenous kappa chains. For example,endogenous kappa chain expression is substantially inactivated such thatsubstantially none of the kappa chain repertoire of the vertebrate(mammal) is provided by endogenous kappa chains. For example, in anembodiment, endogenous heavy chain expression is substantiallyinactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99%of the lambda chain repertoire of the vertebrate (mammal) is provided byendogenous lambda chains. For example, endogenous lambda chainexpression is substantially inactivated such that substantially none ofthe lambda chain repertoire of the vertebrate (mammal) is provided byendogenous lambda chains.

In one aspect this is achieved by inversion of all or part of thenon-human mammal VDJ region, or VJ region, optionally by insertion ofone or more site specific recombinase sites into the genome and then useof these sites in recombinase-mediated excision or inversion of all or apart of the non-human mammal Ig locus. In one aspect a double inversion,may be employed, the first to move the V(D)Js away from the endogenouslocus and then a more local inversion which puts them in the correctorientation. In one aspect a single IoxP site is used to invert thenon-human mammal VDJ region to a centromeric locus or telomeric locus.

In one example, a mouse or mouse cell of the invention comprisesinverted endogenous heavy chain gene segments (eg, VH, D and JH, such asthe entire endogenous heavy chain VDJ region) that are immediately 3′ ofposition 119753123, 119659458 or 120918606 on an endogenous mousechromosome 12. Optionally, the genome of the mouse or cell is homozygousfor said chromosome 12.

The invention also provides:

A cassette for inversion and inactivation of endogenous non-humanvertebrate (eg, mouse or rat) antibody chain gene segments, the segmentsbeing part of an antibody chain locus sequence on a chromosome of anon-human vertebrate (eg, mouse or rat) cell (eg, ES cell) wherein thesequence is flanked at its 3′ end by a site-specific recombination site(eg, lox, rox or frt), the cassette comprising a nucleotide sequenceencoding an expressible label or selectable marker and a compatiblesite-specific recombination site (eg, lox, rox or frt) flanked by a 5′and a 3′ homology arm, wherein the homology arms correspond to or arehomologous to adjacent stretches of sequence in the cell genome on adifferent chromosome or on said chromosome at least 10, 15, 20, 25, 30,35, 40, 45 or 50 mb away from the endogenous gene segments.

The invention also provides:

A cassette for inversion and inactivation of endogenous mouse antibodyheavy chain gene segments, the segments being part of a heavy chainlocus sequence on chromosome 12 of a mouse cell (eg, ES cell) whereinthe sequence is flanked at its 3′ end by a site-specific recombinationsite (eg, lox, rox or frt), the cassette comprising a nucleotidesequence encoding an expressible label or selectable marker and acompatible site-specific recombination site (eg, lox, rox or frt)flanked by a 5′ and a 3′ homology arm, wherein the homology armscorrespond to or are homologous to adjacent stretches of sequence in themouse cell genome on a different chromosome or on chromosome 12 at least10, 15, 20, 25, 30, 35, 40, 45 or 50 mb away from the endogenous genesegments.

The invention provides:

A cassette for inversion and inactivation of endogenous mouse antibodyheavy chain gene segments, the segments being part of a heavy chainlocus sequence on chromosome 12 of a mouse cell (eg, ES cell) whereinthe sequence is flanked at its 3′ end by a site-specific recombinationsite (eg, lox, rox or frt), the cassette comprising a nucleotidesequence encoding an expressible label or selectable marker and acompatible site-specific recombination site (eg, lox, rox or frt)flanked by a 5′ and a 3′ homology arm, wherein (i) the 5′ homology armis mouse chromosome 12 DNA from coordinate 119753124 to coordinate119757104 and the 3′ homology arm is mouse chromosome 12 DNA fromcoordinate 119749288 to 119753123; or (ii) the 5′ homology arm is mousechromosome 12 DNA from coordinate 119659459 to coordinate 119663126 andthe 3′ homology arm is mouse chromosome 12 DNA from coordinate 119656536to 119659458; or (iii) the 5′ homology arm is mouse chromosome 12 DNAfrom coordinate 120918607 to coordinate 120921930 and the 3′ homologyarm is mouse chromosome 12 DNA from coordinate 120915475 to 120918606.

-   -   Embodiment (i) results in an inversion of mouse chromosome 12        from coordinate 119753123 to coordinate 114666436.    -   Embodiment (ii) results in an inversion of mouse chromosome 12        from coordinate 119659458 to coordinate 114666436    -   Embodiment (iii) results in an inversion of mouse chromosome 12        from coordinate 12091806 to coordinate 114666436.

Thus, the invention provides a mouse or mouse cell whose genomecomprises an inversion of a chromosome 12, wherein the inversioncomprises inverted endogenous heavy chain gene segments (eg, VH, D andJH, such as the entire endogenous heavy chain VDJ region); wherein themouse comprises a transgenic heavy chain locus comprising a plurality ofhuman VH gene segments, a plurality of human D segments and a pluralityof human JH segments operably connected upstream of an endogenousconstant region (eg, C mu) so that the mouse or cell (optionallyfollowing differentiation into a B-cell) is capable of expressing anantibody comprising a variable region comprising sequences derived fromthe human gene segments; and wherein the inversion is (i) an inversionof mouse chromosome 12 from coordinate 119753123 to coordinate114666436; (ii) an inversion of mouse chromosome 12 from coordinate119659458 to coordinate 114666436; or (iii) an inversion of mousechromosome 12 from coordinate 12091806 to coordinate 114666436.

In one embodiment, the endogenous gene segments are from a 129-derivedmouse cell (eg, segments from an AB2.1 cell) and the homology arms areisogenic DNA (ie, identical to 129-derived endogenous sequencesdemarcated by the respective coordinates stated in (i) to (iii) above).Thus, no new sequence is created by homologous recombination using thesehomology arms. In another embodiment, the arms are from a mouse strainthat is different from the endogenous strain. The site-specificrecombination sites are mutually compatible and mutually inverted suchthat, on expression of an associated recombinase enzyme (eg, Cre, Dre orFlp), recombination between the site in the inserted inversion cassetteand the site flanking the endogenous gene segments is carried out,thereby inverting and moving the endogenous gene segments far upstream(5′) of their original location in the heavy chain locus. Thisinactivates endogenous heavy chain expression. Similarly, light chaininactivation can be performed by choosing the homology arms of theinversion cassette with reference to a chromosomal region spaced atleast 10, 15, 20, 25, 30, 35, 40, 45 or 50 mb away from the endogenouslight chain locus, the latter comprising a site-specific recombinationsite that is compatible with the site in the inversion cassette.

In one embodiment, the expressible label is a fluorescent label, eg, GFPor a variant thereof (eg, YFP, CFP or RFP). Thus, a label is usedinstead of a selection marker, such as one that confers resistance toallow for selection of transformants.

The invention provides a method of inactivating gene segments of anendogenous antibody locus, the method comprising

-   -   (i) Providing a non-human vertebrate cell (eg, an ES cell, eg, a        mouse ES cell) whose genome comprises an antibody chain locus        comprising endogenous variable region gene segments;    -   (ii) Targeting a site-specific recombination site to flank the        3′ of the 3′-most of said endogenous gene segments;    -   (iii) Targeting a second site-specific recombination site at        least 10 mb away from said endogenous gene segments, the second        site being compatible with the first site inverted with respect        to the first site;    -   (iv) Expressing a recombinase compatible with said sites to        effect site-specific recombination between said sites, thereby        inverting and moving said gene segments away from said locus,        wherein the endogenous gene segments are inactivated; and    -   (v) Optionally developing the cell into a progeny cell or        vertebrate (eg, mouse or rat) whose genome is homozygous for the        inversion.

The genome of the progeny cell or vertebrate can comprise transgenicheavy and/or light chain loci, each capable of expressing antibodychains comprising human variable regions. Optionally, endogenous heavyand kappa light chain expression is inactivated by inverting endogenousheavy and kappa variable region gene segments according to the method ofthe invention. Optionally, endogenous lambda chain expression is alsoinactivated in this way.

In an alternative to the method and inversion cassettes of theinvention, instead of inverting and moving variable region gene segmentsonly, other parts of the endogenous locus can alternatively oradditionally be inverted and moved to effect inactivation. For example,one or more endogenous regulatory elements (eg, Smu and/or Emu) and/orone or more endogenous constant regions (eg, Cmu and/or Cgamma) can beinverted and moved.

Sites that “flank” in the above contexts of the invention can beprovided such that a site-specific recombination site immediately flanksthe endogenous sequence or is spaced therefrom, eg, by no more than 250,200, 250, 100, 50 or 20 kb in the 3′ direction.

In one aspect the non-human mammal genome into which human DNA isinserted comprises endogenous V, (D) and J regions, and the endogenoussequences have not been deleted.

The invention comprises a method for insertion of multiple DNA fragmentsinto a DNA target, suitably to form a contiguous insertion in which theinserted fragments are joined together directly without interveningsequences. The method is especially applicable to the insertion of alarge DNA fragment into a host chromosome which can be carried out in astepwise fashion.

In one aspect the method comprises insertion of a first DNA sequenceinto a target, the sequence having a DNA vector portion and a firstsequence of interest (X1); insertion of a second DNA sequence into thevector portion of the first sequence, the second DNA sequence having asecond sequence of interest (X2) and a second vector portion; and thenexcising any vector sequence DNA separating X1 and X2 to provide acontiguous X1X2, or X2X1 sequence within the target. There is optionallyinsertion of a further one or more DNA sequences, each DNA sequencehaving a further sequence of interest (X3, . . . ) and a further vectorportion, into the vector portion of the preceding DNA sequence, to buildup a contiguous DNA fragment in the target.

The DNA target for insertion of the first DNA sequence may be a specificsite or any point in the genome of a particular cell.

The general method is described herein in relation to the insertion ofelements of the human VDJ region, but is applicable to insertion of anyDNA region, from any organism, and in particular insertion of large DNAfragments of >100 kB, such as 100-250 kb, or even larger, such as thatof the TCR or HLA. Features and approaches described herein in respectof the VDJ insertion may be equally applied to the any of the methodsdisclosed

In one aspect the inserted DNA is human DNA, such as the human VDJ or VJregion, is built up in the genome of a cell, such as an ES cell, in astepwise manner using 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20or more separate insertions for each heavy chain or light chain region.Fragments are suitably inserted at the same or substantially the samecell locus, e.g. ES cell locus, one after another, to form the completeVDJ or VJ region, or part thereof. The present invention also relates tocells and non-human animals comprising intermediates in the processwhose genomes may comprise only a partial VDJ region, such as only humanvariable region DNA.

In a further aspect the method for producing a transgenic non-humanmammal comprises the insertion of human VDJ or VJ regions upstream ofthe host non-human mammal constant region by step-wise insertion ofmultiple fragments by homologous recombination, preferably using aniterative process. Suitably fragments of approximately 100 KB from thehuman VDJ and VJ locus are inserted, suitably to form part of, or acomplete, VDJ or VJ region after the final iteration of the insertionprocess, as disclosed herein.

In one aspect the insertion process commences at a site where aninitiation cassette has been inserted into the genome of a cell, such asan ES cell, providing a unique targeting region. In one aspect theinitiation cassette is inserted in the non-human mammal heavy chainlocus, for use in insertion of human heavy chain DNA. Similarly aninitiation cassette may be inserted in the non-human mammal light chainlocus, for use in insertion of human light chain VJ DNA The initiationcassette suitably comprises a vector backbone sequence with which avector having a human DNA fragment in the same backbone sequence canrecombine to insert the human DNA into the cell (e.g. ES) cell genome,and suitably a selection marker, such as a negative selection marker.Suitably the vector backbone sequence is that of a BAC library, to allowBACs to be used in the construction of the ES cells and mammals. Thevector backbone sequence may however be any sequence which serves as atarget site into which a homologous sequence can insert, for example byhomologous recombination, for example RMCE, and is preferably not DNAencoding any of the VDJ or constant region.

In one aspect the insertion of the first DNA fragment into an initiationcassette is followed by insertion of a second DNA fragment into aportion of the first DNA fragment, suitably a part of the vectorbackbone of the second DNA fragment. In one aspect an inserted DNAfragment comprises a part of the human VDJ region flanked by 5′ and/or3′ sequences that are not from the human VDJ region. In one aspect the5′ and/or 3′ flanking sequences may each contain one or more selectablemarkers, or be capable of creating a selectable system once insertedinto the genome. In one aspect one or both flanking sequences may beremoved from the genome in vitro, or in vivo, following insertion. Inone aspect the method comprises insertion of a DNA fragment followed byselection of both 5′ and 3′ ends of the inserted fragment flanking thehuman VDJ DNA. In one aspect the iterative insertion is made byinsertion of DNA fragments at the 5′ end of the previous insertedfragment, and in this aspect there may be deletion in vivo of the vectorDNA which separates the inserted human DNA sequences, to provide acontiguous human DNA sequence.

In one aspect insertion of human VDJ DNA into a genome may be achievedwithout leaving any flanking DNA in the genome, for example bytransposase mediate DNA excision. One suitable transposase is thePiggybac transposase.

In one aspect the first human variable region fragment is inserted byhomologous recombination at the initiation cassette backbone sequenceand then the DNA of any negative selection marker and initiationcassette are subsequently removed by recombination between recombinasetarget sequences, such as FRT using in this example, FLPase expression.Generally repeated targeted insertions at the (e.g. BAC) backboneinitiation sequence and subsequent removal by rearrangement betweenrecombinase target sequences are repeated to build up the entire humanVDJ region upstream of the host non-mammal constant region.

In one aspect a selectable marker or system may be used in the method.The marker may be generated upon insertion of a DNA fragment into agenome, for example forming a selectable marker in conjunction with aDNA element already present in the genome.

In one aspect the cell (e.g. ES) cell genome does not contain 2identical selectable markers at the same time during the process. It canbe seen that the iterative process of insertion and selection can becarried out using only 2 different selection markers, as disclosed inthe examples herein, and for example the third selectable marker may beidentical to the first marker, as by the time of insertion of the thirdvector fragment the first vector fragment and the first marker has beenremoved.

In one aspect a correct insertion event, is confirmed before moving tothe next step of any multistep cloning process, for example byconfirmation of BAC structure using high density genomic arrays toscreen ES cells to identify those with intact BAC insertions, sequencingand PCR verification.

Initiation Cassette (Also Called a “Landing Pad”)

The invention also relates to a polynucleotide ‘landing pad’ sequence,the polynucleotide comprising nucleic acid regions homologous to regionsof a target chromosome to allow for insertion by homologousrecombination into the target chromosome, and comprising a nucleic acidsite which permits recombinase-driven insertion of nucleic acid into thelanding pad. The invention also relates to vectors, cells and mammals ofthe invention comprising a landing pad as disclosed herein inserted intothe genome of the cell.

The landing pad optionally comprises a non-endogenous S-mu, e.g. a ratS-mu switch

The landing pad optionally comprises (in 5′ to 3′ orientation) a mouseEμ sequence, a non-human, non-mouse (e.g. rat) Switch p and at least aportion of a mouse Cμ or the entire mouse Cμ.

The rat switch sequence optionally comprises or consists of SEQ ID NO 1.

The landing pad optionally comprises the 5′ homology arm of SEQ ID NO 6.

The landing pad optionally has the sequence of SEQ ID 2 or SEQ ID NO 3.

In one embodiment, the landing pad comprises an expressible label. Forexample the label is a fluorescent label, eg, GFP or a variant thereof(eg, YFP, CFP or RFP). Thus, a label is used instead of a selectionmarker (such as one that confers resistance to allow for selection oftransformants).

In an embodiment, the landing pad comprises 5′ and 3′ homology arms forinsertion into the cell genome using homologous recombination. Thehomology arms can be isogenic DNA (eg, identical to 129-derivedendogenous sequences of when a 129-derived ES cell is used). Thus, nonew sequence is created by homologous recombination using these homologyarms. In another embodiment, the arms are from a mouse strain that isdifferent from the endogenous strain (ES cell strain).

The methods of the invention include methods wherein the landing padsequence comprises any of the configurations or sequences as disclosedherein.

Another method of the invention comprises the step of insertion of thelanding pad into a mouse chromosome by homologous recombination betweenmouse J1-4 and mouse C mu sequences.

Another method of the invention comprises the step of insertion of thelanding pad into the mouse chromosome 12 by homologous recombinationbetween mouse J1-4 and E mu.

In one aspect the method uses site specific recombination for insertionof one or more vectors into the genome of a cell, such as an ES cell.Site specific recombinase systems are well known in the art and mayinclude Cre-lox, and FLP/FRT or combinations thereof, in whichrecombination occurs between 2 sites having sequence homology.

Additionally or alternatively to any particular Cre/Lox or FLP/FRTsystem described herein, other recombinases and sites that may be usedin the present invention include Dre recombinase, rox sites, and PhiC31recombinase.

Suitable BACs are available from the Sanger centre, see “A genome-wide,end-sequenced 129Sv BAC library resource for targeting vectorconstruction”. Adams D J, Quail M A, Cox T, van der Weyden L, Gorick BD, Su Q, Chan W I, Davies R, Bonfield J K, Law F, Humphray S, Plumb B,Liu P, Rogers J, Bradley A. Genomics. 2005 December; 86(6):753-8. Epub2005 Oct. 27. The Wellcome Trust Sanger Institute, Hinxton,Cambridgeshire CB10 1SA, UK. BACs containing human DNA are alsoavailable from, for example, Invitrogen™. A suitable library isdescribed in Osoegawa K et al, Genome Research 2001. 11: 483-496.

In one aspect a method of the invention specifically comprises:

-   -   (1) insertion of a first DNA fragment into a non-human ES cell,        the fragment containing a first portion of human VDJ or VJ        region DNA and a first vector portion containing a first        selectable marker;    -   (2) optionally deletion of the a part of the first vector        portion;    -   (3) insertion of a second DNA fragment into a non-human ES cell        containing the first DNA fragment, the insertion occurring        within the first vector portion, the second DNA fragment        containing a second portion of the human VDJ or VJ region and a        second vector portion containing a second selectable marker, (4)        deletion of the first selectable marker and first vector        portion, preferably by a recombinase enzyme action;    -   (5) insertion of a third DNA fragment into a non-human ES cell        containing the second DNA fragment, the insertion occurring        within the second vector portion, the third DNA fragment        containing a third portion of the human VDJ or VJ region and a        third vector portion containing third selectable marker,    -   (6) deletion of the second selectable marker and second vector        portion; and    -   (7) iteration of the steps of insertion and deletion, as        necessary, for fourth and further fragments of the human VDJ or        VJ human regions, as necessary, to produce an ES cell with a        part or all of the human VDJ or VJ region inserted as disclosed        herein, and suitably to remove all the vector portions within        the ES cell genome.

In another aspect the invention comprises

-   -   (1) insertion of DNA forming an initiation cassette into the        genome of a cell;    -   (2) insertion of a first DNA fragment into the initiation        cassette, the first DNA fragment comprising a first portion of a        human DNA and a first vector portion containing a first        selectable marker or generating a selectable marker upon        insertion;    -   (3) optionally removal of part of the vector DNA    -   (4) insertion of a second DNA fragment into the vector portion        of the first DNA fragment, the second DNA fragment containing a        second portion of human DNA and a second vector portion, the        second vector portion containing a second selectable marker, or        generating a second selectable marker upon insertion;    -   (5) optionally, removal of any vector DNA to allow the first and        second human DNA fragments to form a contiguous sequence; and    -   (6) iteration of the steps of insertion of human VDJ DNA and        vector DNA removal, as necessary, to produce a cell with all or        part of the human VDJ or VJ region sufficient to be capable of        generating a chimaeric antibody in conjunction with a host        constant region, wherein the insertion of one, or more, or all        of the DNA fragments uses site specific recombination.

In one aspect the non-human mammal is able to generate a diversity of atleast 1×10⁶ different functional chimaeric immunoglobulin sequencecombinations.

In one aspect the targeting is carried out in ES cells derived from themouse C57BL/6N, C57BL/6J, 129S5 or 129Sv strain.

In one aspect non-human animals, such as mice, are generated in aRAG-1-deficient or a RAG-2-deficient background, or other suitablegenetic background which prevents the production of mature host B and Tlymphocytes.

In one aspect the non-human mammal is a rodent, suitably a mouse, andcells of the invention, are rodent cells or ES cells, suitably mouse EScells.

The ES cells of the present invention can be used to generate animalsusing techniques well known in the art, which comprise injection of theES cell into a blastocyst followed by implantation of chimaericblastocystys into females to produce offspring which can be bred andselected for homozygous recombinants having the required insertion. Inone aspect the invention relates to a chimeric animal comprised of EScell-derived tissue and host embryo derived tissue. In one aspect theinvention relates to genetically-altered subsequent generation animals,which include animals having a homozygous recombinants for the VDJand/or VJ regions.

In a further aspect the invention relates to a method for producing anantibody specific to a desired antigen the method comprising immunizinga transgenic non-human mammal as above with the desired antigen andrecovering the antibody (see e.g. Harlow, E. & Lane, D. 1998, 5thedition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press,Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the NationalAcademy of Sciences (2004) 101:257-259). Suitably an immunogenic amountof the antigen is delivered. The invention also relates to a method fordetecting a target antigen comprising detecting an antibody produced asabove with a secondary detection agent which recognises a portion ofthat antibody.

In a further aspect the invention relates to a method for producing afully humanised antibody comprising immunizing a transgenic non-humanmammal as above with the desired antigen, recovering the antibody orcells expressing the antibody, and then replacing the non-human mammalconstant region with a human constant region. This can be done bystandard cloning techniques at the DNA level to replace the non-humanmammal constant region with an appropriate human constant region DNAsequence—see e.g. Sambrook, J and Russell, D. (2001, 3'd edition)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, N.Y.).

In a further aspect the invention relates to humanised antibodies andantibody chains produced according to the present invention, both inchimaeric and fully humanised form, and use of said antibodies inmedicine. The invention also relates to a pharmaceutical compositioncomprising such an antibodies and a pharmaceutically acceptable carrieror other excipient.

Antibody chains containing human sequences, such as chimaerichuman-non-human antibody chains, are considered humanised herein byvirtue of the presence of the human protein coding regions region. Fullyhumanised antibodies may be produced starting from DNA encoding achimaeric antibody chain of the invention using standard techniques.

Methods for the generation of both monoclonal and polyclonal antibodiesare well known in the art, and the present invention relates to bothpolyclonal and monoclonal antibodies of chimaeric or fully humanisedantibodies produced in response to antigen challenge in non-humanmammals of the present invention.

In a yet further aspect, chimaeric antibodies or antibody chainsgenerated in the present invention may be manipulated, suitably at theDNA level, to generate molecules with antibody-like properties orstructure, such as a human variable region from a heavy or light chainabsent a constant region, for example a domain antibody; or a humanvariable region with any constant region from either heavy or lightchain from the same or different species; or a human variable regionwith a non-naturally occurring constant region; or human variable regiontogether with any other fusion partner. The invention relates to allsuch chimaeric antibody derivatives derived from chimaeric antibodiesidentified according to the present invention.

In a further aspect, the invention relates to use of animals of thepresent invention in the analysis of the likely effects of drugs andvaccines in the context of a quasi-human antibody repertoire.

The invention also relates to a method for identification or validationof a drug or vaccine, the method comprising delivering the vaccine ordrug to a mammal of the invention and monitoring one or more of: theimmune response, the safety profile; the effect on disease.

The invention also relates to a kit comprising an antibody or antibodyderivative as disclosed herein and either instructions for use of suchantibody or a suitable laboratory reagent, such as a buffer, antibodydetection reagent.

The invention also relates to a method for making an antibody, or partthereof, the method comprising providing:

-   -   (i) a nucleic acid encoding an antibody, or a part thereof,        obtained according to the present invention; or    -   (ii) sequence information from which a nucleic acid encoding an        antibody obtained according to the present invention, or part        thereof, can be expressed to allow an antibody to be produced.

The present invention also relates to a chimaeric antibody comprising ahuman variable region and a non-human vertebrate or mammal (optionally arat or mouse) constant region (optionally a C gamma or C mu), whereinthe antibody is encoded by a nucleotide sequence corresponding to thenucleotide sequence of a chimaeric heavy chain locus of a cell(optionally a B-cell, ES cell or hybridoma), the locus comprising anon-human vertebrate constant region nucleotide sequence and arearranged VDJ nucleotide sequence produced by the in vivo rearrangementof a human V region, a human D region and a human J region, the V regionbeing selected from one of a V1-3 region, V2-5 region, V4-4 region, V1-2region or V6-1 region, and optionally a V1-3 or V6-1 segment.Optionally, the J region is any of JH1, JH2, JH3, JH4, JH5 or JH6, andin one aspect is JH4 or JH6. The D region is, in one aspect, any D3-9,D3-10, D6-13 or D6-19. In one example, rearranged VDJ nucleotidesequence is produced by the in vivo rearrangement of human V1-3 and JH4(optionally with D3-9, D3-10, D6-13 or D-19); or V1-3 and JH6(optionally with D3-9, D3-10, D6-13 or D-19); or V6-1 and JH4(optionally with D3-9, D3-10, D6-13 or D-19); or V6-1 and JH6(optionally with D3-9, D3-10, D6-13 or D-19). In one example therearranged VDJ nucleotide sequence is produced by the in vivorearrangement of human V6-1 DH3-10, V1-3 DH3-10, V1-3 DH6-19, V1-3 Dh3-9or V6-1 DH6-19. In one aspect the antibody comprises any combinationexemplified in the Examples and Figures herein. Optionally, the in vivorearrangement is in a cell (eg, B cell or ES cell) derived from the samenon-human vertebrate species as the constant region sequence (eg, amouse B cell or ES cell). The invention also relates to a non-humanvertebrate or mammal cell (eg, a B-cell or ES cell or hybridoma) whosegenome comprises a chimaeric heavy chain locus as described above inthis paragraph. The invention also relates to a non-human vertebrate ormammal (eg, a mouse or rat) whose genome comprises a chimaeric heavychain locus as described above in this paragraph.

The present invention also relates to a non-human vertebrate or mammalhaving a genome encoding a chimaeric antibody, the chimaeric antibodycomprising a human variable region and a non-human vertebrate or mammal(optionally a rat or mouse) constant region (optionally a C gamma or Cmu), the mammal:

-   -   expressing more V1-3 antibodies than V2-5, V4-4, V1-2 or V6-1        antibodies; and/or    -   expressing more V1-3 JH4 or V1-3 JH6 antibodies than any of,        individually, V1-3 JH1, V1-3 JH2, V1-3 JH3 or V1-3 JH5        antibodies, and/or    -   expressing more V6-1 JH4 or V6-1 JH6 antibodies than any of,        individually, V6-1 JH1, V6-1 JH2, V6-1 JH3 or V6-1 JH5        antibodies and/or    -   expressing a greater number of V1-3 DH3-10 antibodies than        antibodies V1-3 with any other D region. Expression of        antibodies can be assessed by methods readily available to the        skilled person and as conventional in the art. For example,        expression can be assessed at the mRNA level as shown in the        examples below.

The invention also relates to a chimaeric antibody comprising a humanvariable region and a non-human vertebrate or mammal (optionally a rator mouse) constant region (optionally a light chain constant region),wherein the antibody is obtainable from a mammal (optionally a rat ormouse) whose genome comprises an antibody chain locus comprising agermline human kappa V1-8 and germline human kappa J1 sequence, andwherein the antibody is obtainable by in vivo recombination in saidmammal of the V1-8 and J1 sequences and wherein the antibody has avariable region sequence which is different from that which is encodedby germline human kappa V1-8 and germline human kappa J1 sequences.Thus, in this aspect of the invention the human germline sequences areable to undergo productive rearrangement to form a coding sequencewhich, in conjunction with the non-human constant region sequence, canbe expressed as a chimaeric antibody chain having at least a completehuman variable region and a non-human constant region. This is incontrast (as the examples show below) to the combination of the germlinehuman kappa V1-8 and germline human kappa J1 sequences per se, which donot provide for an antibody coding sequence (due to the inclusion ofstop codons). In one aspect the rearranged sequence of the chimaericantibody is a result of somatic hypermutation. In one aspect theantibody is a kappa antibody; in another aspect the antibody comprises anon-human heavy chain constant region (eg, a rat or mouse C gamma or Cmu). The antibody sequence optionally comprises a X₁X₂ T F G Q, whereX₁X₂=PR, RT, or PW (SEQ ID No 21); optionally a X₁X₂ T F G Q G T K V E IK R A D A (SEQ ID No 22) motif. Such motifs are not found in theequivalent position in the germline sequence as shown in the examples.The invention also relates to a non-human vertebrate or mammal cell (eg,a B-cell or ES cell or hybridoma) whose genome comprises a chimaericantibody chain locus as described above in this paragraph. The inventionalso relates to a non-human vertebrate or mammal (eg, a mouse or rat)whose genome comprises a chimaeric antibody chain locus as describedabove in this paragraph.

The invention also relates to a chimaeric antibody comprising a humanvariable region and a non-human vertebrate or mammal (optionally a rator mouse) constant region (optionally a light chain constant region),wherein the antibody is obtainable from a mammal (optionally a rat ormouse) whose genome comprises an antibody chain locus comprising agermline human kappa V1-6 and germline human kappa J1 sequence, andwherein the antibody is obtainable by in vivo recombination in saidmammal of the V1-6 and J1 sequences and wherein the antibody has avariable region sequence which is different from that which is encodedby germline human kappa V1-6 and germline human kappa J1 sequences.Thus, in this aspect of the invention the human germline sequences areable to undergo productive rearrangement to form a coding sequencewhich, in conjunction with the non-human constant region sequence, canbe expressed as a chimaeric antibody chain having at least a completehuman variable region and a non-human constant region. This is incontrast (as the examples show below) to the combination of the germlinehuman kappa V1-6 and germline human kappa J1 sequences per se, which donot provide for an antibody coding sequence (due to the inclusion ofstop codons). In one aspect the rearranged sequence of the chimaericantibody is a result of somatic hypermutation. In one aspect theantibody is a kappa antibody; in another aspect the antibody comprises anon-human heavy chain constant region (eg, a rat or mouse C gamma or Cmu). The antibody sequence optionally comprises a X₃X₄ T F G Q, whereX₃X₄=PR or PW (SEQ ID No 23); optionally a X₃X₄ T F G Q G T K V E I K RA D A (SEQ ID No 24) motif. Such motifs are not found in the equivalentposition in the germline sequence as shown in the examples. Theinvention also relates to a non-human vertebrate or mammal cell (eg, aB-cell or ES cell or hybridoma) whose genome comprises a chimaericantibody chain locus as described above in this paragraph. The inventionalso relates to a non-human vertebrate or mammal (eg, a mouse or rat)whose genome comprises a chimaeric antibody chain locus as describedabove in this paragraph.

The invention also relates to a chimaeric antibody comprising a humanvariable region and a non-human (optionally a rat or mouse) constantregion (optionally a C gamma or C mu or a C kappa), wherein the antibodyis obtainable from a mammal (optionally a rat or mouse) whose genomecomprises an antibody chain locus comprising a germline human kappa V1-5and germline human kappa J1 sequence, and wherein the antibody isobtainable by in vivo recombination in said mammal of the V1-5 and J1sequences. The invention also relates to a non-human vertebrate ormammal cell (eg, a B-cell or ES cell or hybridoma) whose genomecomprises a chimaeric antibody chain locus as described above in thisparagraph. The invention also relates to a non-human vertebrate ormammal (eg, a mouse or rat) whose genome comprises a chimaeric antibodychain locus as described above in this paragraph.

The invention also relates to a chimaeric antibody comprising a humanvariable region and a non-human (optionally a rat or mouse) constantregion (optionally a C gamma or C mu or a C kappa), wherein the antibodyis obtainable from a mammal (optionally a rat or mouse) whose genomecomprises an antibody chain locus comprising a germline human kappa V1-5and germline human kappa J4 sequence, and wherein the antibody isobtainable by in vivo recombination in said mammal of the V1-5 and J4sequences. The invention also relates to a non-human vertebrate ormammal cell (eg, a B-cell or ES cell or hybridoma) whose genomecomprises a chimaeric antibody chain locus as described above in thisparagraph. The invention also relates to a non-human vertebrate ormammal (eg, a mouse or rat) whose genome comprises a chimaeric antibodychain locus as described above in this paragraph.

Antibodies of the invention may be isolated, in one aspect beingisolated from the cell or organism in which they are expressed.

A non-human mammal whose genome comprises:

-   -   (a) the human IgH VDJ region upstream of the host non-human        mammal constant region; and    -   (b) the human Ig light chain kappa V and J regions upstream of        the host non-human mammal kappa constant region and/or the human        Ig light chain lambda V and J regions upstream of the host        non-human mammal lambda constant region;

wherein the non-human mammal is able to produce a repertoire ofchimaeric antibodies having a non-human mammal constant region and ahuman variable region,

and optionally wherein the non-human mammal genome is modified toprevent expression of fully host-species specific antibodies.

A non-human mammal ES cell whose genome comprises:

-   -   (a) the human IgH V, D and J region upstream of a non-human        mammal constant region; and    -   (b) the human Ig locus light chain kappa V and J regions        upstream of the host non-human mammal kappa constant region,        and/or the human Ig locus light chain lambda V and J regions        upstream of the host non-human mammal lambda constant region

wherein the ES cell is capable of developing into a non-human mammal,being able to produce a repertoire of antibodies which are chimaeric,having a non-human mammal constant region and a human variable region.

A method for producing a transgenic non-human mammal able to produce arepertoire of chimaeric antibodies, the antibodies having a non-humanmammal constant region and a human variable region, the methodcomprising inserting by homologous recombination into a non-human mammalES cell genome

-   -   (a) the human IgH VDJ region upstream of the host non-human        mammal heavy chain constant region, and    -   (b) the human IgL VJ region for lambda or kappa chains upstream        of the host non-human mammal lambda or kappa chain constant        region, respectively

such that the non-human mammal is able to produce a repertoire ofchimaeric antibodies having a non-human mammal constant region and ahuman variable region, wherein steps (a) and (b) can be carried out ineither order and each of steps (a) and (b) can be carried out in astepwise manner or as a single step.

In one aspect the insertion of human VDJ or VJ regions upstream of thehost non-human mammal constant region is accomplished by step-wiseinsertion of multiple fragments by homologous recombination.

In one aspect the step-wise insertions commence at a site where aninitiation cassette has been inserted into the genome of an ES cellproviding a unique targeting region consisting of a BAC backbonesequence and a negative selection marker.

In one aspect the first human variable region fragment is inserted byhomologous recombination at the initiation cassette BAC backbonesequence and said negative selection marker and initiation cassette aresubsequently removed by recombination between recombinase targetsequences.

In one aspect repeated targeted insertions at the BAC backboneinitiation sequence and subsequent removal of the backbone byrearrangement between recombinase target sequences is repeated to buildup the entire human VDJ region upstream of the host non-mammal constantregion.

Insertion of Human Variable Region Gene Segments Precisely within theEndogenous Mouse JH4-Cmu Intron

There is further provided a cell or non human mammal according to theinvention wherein the mammal is a mouse or the cell is a mouse cell andwherein the insertion of the human heavy chain DNA is made in a mousegenome between coordinates 114,667,091 and 114,665,190 of mousechromosome 12.

There is further provided a cell or non human mammal according to theinvention wherein the insertion of the human heavy chain DNA is made atcoordinate 114,667,091.

There is further provided a cell or non human mammal according to theinvention wherein the human IgH VDJ region comprises nucleotides105,400,051 to 106,368,585 from human chromosome 14 (coordinates referto NCBI36 for the human genome).

There is further provided a method, cell or non human mammal accordingto the invention wherein a human coding region DNA sequence is in afunctional arrangement with a non-human mammal control sequence, suchthat transcription of the human DNA is controlled by the non-humanmammal control sequence. In one example, the initiation cassette isinserted between the mouse J4 and C alpha exons. There is furtherprovided an initiation cassette suitable for use in the methodcomprising a vector backbone sequence and a selection marker.

The invention provides the following aspects (starting at aspect number103):

-   103. A cell or non human mammal according to any one of the above    configurations, examples, embodiments or aspects, wherein the mammal    is a mouse or the cell is a mouse cell and wherein the insertion of    the human heavy chain DNA is made in a mouse genome between    coordinates 114,667,091 and 114,665,190 of mouse chromosome 12.-   104. A cell or non human mammal according to any one of the above    configurations, examples, embodiments or aspects, wherein the    insertion of the human heavy chain DNA is made at coordinate    114,667,091.-   105. A cell or mammal according to any one of the above    configurations, examples, embodiments or aspects, wherein the human    IgH VDJ region comprises nucleotides 105,400,051 to 106,368,585 from    human chromosome 14 (coordinates refer to NCBI36 for the human    genome).-   106. A method, cell or mammal according to any one of the above    configurations, examples, embodiments or aspects, wherein a human    coding region DNA sequence is in a functional arrangement with a    non-human mammal control sequence, such that transcription of the    human DNA is controlled by the non-human mammal control sequence.-   107. A method according to aspect 106 wherein the initiation    cassette is inserted between the mouse J4 and C alpha exons.-   108. An initiation cassette suitable for use in the method of aspect    107 comprising a vector backbone sequence and a selection marker.

Inactivation of Endogenous Antibody Chain Expression by Insertion ofHuman Antibody Variable Region Gene Segments

-   109. A non-human vertebrate (optionally a mouse or rat) or non-human    vertebrate cell (optionally a mouse or rat cell) having a genome    that    -   (i) comprises a transgenic antibody chain locus capable of        expressing an antibody chain comprising a human variable region        (optionally following antibody gene rearrangement); and    -   (ii) is inactivated for endogenous non-human vertebrate antibody        chain expression; wherein the transgenic locus comprises    -   (iii) a DNA sequence comprising a plurality of human antibody        variable region gene segments inserted between endogenous        antibody variable region gene segments and an endogenous        antibody constant region, whereby endogenous antibody chain        expression is inactivated.

The transgenic locus is a heavy chain or light chain locus.

Inactivation of endogenous heavy chain expression in non-humanvertebrates such as mice and rats has involved the deletion of all orpart of the endogenous heavy chain VDJ region (including sequencesbetween gene segments). The ADAM6 genes are present in the endogenousmouse VDJ region. In mouse, there are two copies of ADAM6 (ADAM6a,ADAM6b) located between the VH and D gene segments in the IgH locus ofchromosome 12 (in the intervening region between mouse VH5-1 and D1-1gene segments). These two adjacent intronless ADAM6 genes have 95%nucleotide sequence identity and 90% amino acid identity. In human andrat, there is only one ADAM6 gene. Expression pattern analysis of mouseADAM6 shows that it is exclusively expressed in testis [1]. AlthoughADAM6 transcripts can be detected in lymphocytes, it is restricted tothe nucleus, suggesting that the transcription of ADAM6 gene inparticular was due to transcriptional read-through from the D regionrather than active messenger RNA production [2]. In rat, ADAM6 is onchromosome 6.

Mature ADAM6 protein is located on the acrosome and the posteriorregions of sperm head. Notably, ADAM6 forms a complex with ADAM2 andADAM3, which is required for fertilization in mice [3]. Reference [4]implicates ADAM6 in a model where this protein interacts with ADAM3after ADAM6 is sulphated by TPST2, sulphation of ADAM6 being criticalfor stability and/or complex formation involving ADAM6 and ADAM3, andthus ADAM6 and ADAM3 are lost from Tpst2-null sperm. The study observesthat Tpst2-deficient mice have male infertility, sperm mobility defectsand possible abnormalities in sperm-egg membrane interactions.

Thus, the maintenance of ADAM6 expression in sperm is crucial forfertility. Thus, it is thought that transgenic male mice and rats inwhich ADAM6 genes have been deleted are not viably fertile. This hampersbreeding of colonies and hampers the utility of such mice as transgenicantibody-generating platforms. It would be desirable to provide improvednon-human transgenic antibody-generating vertebrates that are fertile.

-   [1]. Choi I, et. al., Characterization and comparative genomic    analysis of intronless Adams with testicular gene expression.    Genomics. 2004 April; 83(4):636-46.-   [2]. Featherstone K, Wood A L, Bowen A J, Corcoran A E. The mouse    immunoglobulin heavy chain V-D intergenic sequence contains    insulators that may regulate ordered V(D)J recombination. J Biol    Chem. 2010 Mar. 26; 285(13):9327-38. Epub 2010 Jan. 25.-   [3]. Han C, et. al., Comprehensive analysis of reproductive ADAMs:    relationship of ADAM4 and ADAM6 with an ADAM complex required for    fertilization in mice. Biol Reprod. 2009 May; 80(5):1001-8. Epub    2009 Jan. 7.-   [4]. Marcello et al, Lack of tyrosylprotein sulfotransferase-2    activity results in altered sperm-egg interactions and loss of ADAM3    and ADAM6 in epididymal sperm, J Biol Chem. 2011 Apr. 15;    286(15):13060-70. Epub 2011 Feb. 21.

According to aspect 109 of the invention, inactivation does not involvedeletion of the VDJ region or part thereof including endogenous ADAM6,but instead inactivation by insertion allows for the preservation ofendogenous ADAM6 and thus does not risk infertility problems.

The final mouse resulting from the method (or a mouse derived from acell produced by the method) is in one embodiment a male, so that theinvention improves upon the prior art male transgenic mice that areinfertile as a result of genomic manipulation. Fertile mice producesperm that can fertilise eggs from a female mouse. Fertility is readilydetermined, for example, by successfully breeding to produce an embryoor child mouse. In another embodiment, the method of the invention makesa final female mouse. Such females are, of course, useful for breedingto create male progeny carrying ADAM6 and which are fertile.

In one embodiment of aspect 109, the genome is homozygous for thetransgenic locus. For example, the genome is homozygous for endogenousADAM6 genes.

In one embodiment of the vertebrate of aspect 109, the genome isinactivated for expression of endogenous heavy and kappa (and optionallyalso lambda) chains.

In one embodiment, in part (iii) of aspect 109 said DNA comprises humanVH, D and JH gene segments or human VL and JL gene segments (eg, VK andJK gene segments). In an example, the DNA comprises a landing pad havinga selectable marker, eg, a HPRT gene, neomycin resistance gene or apuromycin resistance gene; and/or a promoter.

In one embodiment, in part (iii) of aspect 109 the endogenous genesegments are the entire endogenous VDJ region of a heavy chain locusand/or the endogenous constant region is a Cmu or Cgamma.

In one embodiment, in part (iii) of aspect 109 the endogenous genesegments are the entire endogenous VJ region of a kappa chain locusand/or the endogenous constant region is a Ckappa

In one embodiment, in part (iii) of aspect 109 the endogenous genesegments are the entire endogenous VJ region of a lambda chain locusand/or the endogenous constant region is a Clambda.

The non-human vertebrate cell can be a hybridoma, B-cell, ES cell or anIPS cell. When the cell is an ES cell or IPS cell, the endogenousantibody chain expression is inactivated following differentiation ofthe cell into a progeny B-cell (eg, in a B-cell in a non-humanvertebrate).

The invention further provides:

-   110. The vertebrate or cell according to aspect 109, wherein said    plurality of human antibody gene segments comprises at least 11    human V segments and/or at least 6 human J segments, eg at least 11    human VH gene segments and at least 6 human JH segments and    optionally also at least 27 human D segments; optionally with the    human inter-gene segment intervening sequences. In an embodiment,    the human antibody gene segments are provided by a stretch of DNA    sequence of human chromosome 14, comprising the gene segments and    intervening sequences in germline configuration.-   111. The vertebrate or cell according to aspect 109 or 110, wherein    said inserted DNA sequence comprises a human nucleotide sequence    comprising said antibody gene segments, wherein the nucleotide    sequence is at least 110, 130, 150, 170, 190, 210, 230, 250, 270 or    290 kb. In an embodiment, the nucleotide sequence corresponds to a    stretch of DNA sequence of human chromosome 14, comprising the gene    segments and intervening sequences in germline configuration, eg, at    least a sequence corresponding to the nucleotide sequence from    coordinate 106328951 to coordinate 106601551 of a human chromosome    14, eg, a sequence in the GRCH37/hg19 sequence database.-   112. The vertebrate or cell according to aspect 109, wherein the    transgenic locus is a light chain kappa locus and the human antibody    gene segments are between the 3′-most endogenous Jk gene segment and    endogenous Ck; optionally wherein the human antibody gene segments    comprise five functional human JA-CA clusters and at least one human    VA gene segment, eg, at least a sequence corresponding to the    nucleotide sequence from coordinate 23217291 to 23327884 of a lambda    locus found on a human chromosome 22.-   113. The vertebrate or cell according to any one of aspects 109 to    112, wherein the transgenic locus is a heavy chain locus and the    human antibody gene segments are between the 3′-most endogenous JH    gene segment (eg, JH4 in a mouse genome) and endogenous Cmu.-   114. The vertebrate or cell according to any one of aspects 109 to    113, wherein the genome is homozygous for said transgenic locus.-   115. A mouse or mouse cell or a rat or rat cell according to any one    of aspects 109 to 114.-   116. A method of making a non-human vertebrate cell (optionally a    mouse or rat cell), the method comprising    -   (a) providing a non-human ES cell whose genome comprises an        endogenous antibody chain locus comprising endogenous antibody        variable region gene segments and an endogenous antibody        constant region; and    -   (b) making a transgenic antibody chain locus by inserting into        said endogenous locus a DNA sequences comprising a plurality of        human antibody variable region gene segments between said        endogenous antibody variable region gene segments and said        endogenous constant region, so that the human antibody variable        region gene segments are operably connected upstream of the        endogenous constant region,    -   whereby a non-human vertebrate ES cell is produced that is        capable of giving rise to a progeny cell in which endogenous        antibody expression is inactivated and wherein the progeny is        capable of expressing antibodies comprising human variable        regions; and    -   (c) optionally differentiating said ES cell into said progeny        cell or a non-human vertebrate (eg, mouse or rat) comprising        said progeny cell.-   117. The method according to aspect 116, wherein said plurality of    human antibody gene segments comprises at least 11 human V segments.-   118. The method according to aspect 116 or 117, wherein said    plurality of human antibody gene segments comprises at least 6 human    J segments.-   119. The method according to aspect 116, 117 or 118, wherein a human    nucleotide sequence is inserted in step (b), the nucleotide sequence    comprising said antibody gene segments, wherein the nucleotide    sequence is at least 110 kb.-   120. The method according to any one of aspects 110 to 113, wherein    the endogenous locus is a heavy chain locus and the human antibody    gene segments are between the 3′-most endogenous JH gene segment and    endogenous Cmu.-   121. The method according to any one of aspects 116 to 120, wherein    the progeny cell is homozygous for said transgenic locus.

In one embodiment of the method of aspect 116, the method comprisesinactivating the genome for expression of endogenous heavy and kappa(and optionally also lambda) chains.

In one embodiment of the method of aspect 116, in part (b) said DNAsequence comprises human VH, D and JH gene segments or human VL and JLgene segments (eg, VK and JK gene segments). In an example, the DNAcomprises a landing pad having a selectable marker, eg, a HPRT gene,neomycin resistance gene or a puromycin resistance gene; and/or apromoter.

In one embodiment, in part (b) of aspect 116 the endogenous genesegments are the entire endogenous VDJ region of a heavy chain locusand/or the endogenous constant region is a Cmu or Cgamma.

In one embodiment, in part (b) of aspect 116 the endogenous genesegments are the entire endogenous VJ region of a kappa chain locusand/or the endogenous constant region is a Ckappa

In one embodiment, in part (b) of aspect 116 the endogenous genesegments are the entire endogenous VJ region of a lambda chain locusand/or the endogenous constant region is a Clambda.

The non-human vertebrate cell can be a hybridoma, B-cell, ES cell or anIPS cell. When the cell is an ES cell or IPS cell, the endogenousantibody chain expression is inactivated following differentiation ofthe cell into a progeny B-cell (eg, in a B-cell in a non-humanvertebrate).

The invention further provides:

The method according to aspect 116, wherein said inserted DNA sequencecomprises a human nucleotide sequence comprising said human antibodygene segments, wherein the nucleotide sequence is at least 110, 130,150, 170, 190, 210, 230, 250, 270 or 290 kb. In an embodiment, thenucleotide sequence corresponds to a stretch of DNA sequence of humanchromosome 14, comprising the gene segments and intervening sequences ingermline configuration, eg, at least a sequence corresponding to thenucleotide sequence from coordinate 106328951 to coordinate 106601551 ofa human chromosome 14, eg, a sequence in the GRCH37/hg19 sequencedatabase.

The method according to aspect 116, wherein the transgenic locus is alight chain kappa locus and the human antibody gene segments are betweenthe 3′-most endogenous Jk gene segment and endogenous Ck; optionallywherein the human antibody gene segments comprise five functional humanJA-CA clusters and at least one human VA gene segment, eg, at least asequence corresponding to the nucleotide sequence from coordinate23217291 to 23327884 of a lambda locus found on a human chromosome 22.

The method according to aspect 116, wherein, wherein the transgeniclocus is a heavy chain locus and the human antibody gene segments areinserted between the 3′-most endogenous JH gene segment (eg, JH4 in amouse genome) and endogenous Cmu.

-   122. The method according to any one of aspects 116 to 121,    comprising making the genome of the progeny homozygous for said    transgenic locus.

Isolating Antibodies from Transgenic Non-Human Vertebrates of theInvention & Useful Antigen-Specific Antibodies ofTherapeutically-Relevant Affinities

-   123. A method of isolating an antibody that binds a predetermined    antigen, the method comprising    -   (a) providing a vertebrate (optionally a mammal; optionally a        mouse or rat according to any one of the above configurations,        examples, embodiments or aspects;    -   (b) immunising said vertebrate with said antigen (optionally        wherein the antigen is an antigen of an infectious disease        pathogen);    -   (c) removing B lymphocytes from the vertebrate and selecting one        or more B lymphocytes expressing antibodies that bind to the        antigen;    -   (d) optionally immortalising said selected B lymphocytes or        progeny thereof, optionally by producing hybridomas therefrom;        and    -   (e) isolating an antibody (eg, and IgG-type antibody) expressed        by the B lymphocytes.-   124. The method of aspect 123, comprising the step of isolating from    said B lymphocytes nucleic acid encoding said antibody that binds    said antigen; optionally exchanging the heavy chain constant region    nucleotide sequence of the antibody with a nucleotide sequence    encoding a human or humanised heavy chain constant region and    optionally affinity maturing the variable region of said antibody;    and optionally inserting said nucleic acid into an expression vector    and optionally a host.-   125. The method of aspect 123 or 124, further comprising making a    mutant or derivative of the antibody produced by the method of    aspect 122 or 123.

As demonstrated by the examples below, the non-human vertebrates of theinvention are able to produce antigen-specific antibodies of sub-50 nMaffinity with human sequences in their CDR3 regions. Thus, the inventionfurther provides:

-   126. An antibody or fragment (eg, a Fab or Fab₂) thereof comprising    variable regions that specifically bind a predetermined antigen with    a sub-50 nM affinity (optionally sub-40, 30, 20, 10, 1, 0.1 or 0.01    nM) as determined by surface plasmon resonance, wherein the antibody    is isolated from a non-human vertebrate (optionally a mammal;    optionally a mouse or rat) according to any one of the above    configurations, examples, embodiments or aspects and comprises heavy    chain CDR3s (as defined by Kabat) encoded by a rearranged VDJ of    said vertebrate, wherein the VDJ is the product of rearrangement in    vivo of a human JH gene segment of a heavy chain locus of said    vertebrate with D (optionally a human D gene segment of said locus)    and VH gene segments.

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the antibody is determined using SPR by

-   1. Coupling anti-mouse (or other relevant non-human vertebrate) IgG    (eg, Biacore BR-1008-38) to a biosensor chip (eg, GLM chip) such as    by primary amine coupling;-   2. Exposing the anti-mouse IgG (non-human vertebrate antibody) to a    test IgG antibody to capture test antibody on the chip;-   3. Passing the test antigen over the chip's capture surface at 1024    nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and-   4. And determining the affinity of binding of test antibody to test    antigen using surface plasmon resonance, eg, under an SPR condition    discussed above (eg, at 25° C. in physiological buffer). SPR can be    carried out using any standard SPR apparatus, such as by Biacore™ or    using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

The invention also relates to an scFv, diabody or other antibodyfragment comprising a VH and VL domain from an antibody or fragment ofaspect 126 (optionally following affinity maturation, eg, by phagedisplay).

In one embodiment, the antigen is a serpin, eg, ovalbumin, antithrombinor antitrypsin. Serpins are a group of proteins with similar structuresthat were first identified as a set of proteins able to inhibitproteases. The acronym serpin was originally coined because many serpinsinhibit chymotrypsin-like serine proteases (serine protease inhibitors).The first members of the serpin superfamily to be extensively studiedwere the human plasma proteins antithrombin and antitrypsin, which playkey roles in controlling blood coagulation and inflammation,respectively. Initially, research focused upon their role in humandisease: antithrombin deficiency results in thrombosis and antitrypsindeficiency causes emphysema. In 1980 Hunt and Dayhoff made thesurprising discovery that both these molecules share significant aminoacid sequence similarity to the major protein in chicken egg white,ovalbumin, and they proposed a new protein superfamily.

-   127. An antibody or fragment that is identical to an antibody of    aspect 126 or a derivative thereof (optionally a derivative whose    constant regions are human and/or an affinity matured derivative)    that specifically binds said antigen with a sub-50 nM affinity as    determined by surface plasmon resonance.-   128. A pharmaceutical composition comprising an antibody or fragment    of aspect 126 or 127 and a pharmaceutically-acceptable diluent,    excipient or carrier.-   129. A nucleotide sequence encoding a heavy chain variable region of    an antibody or fragment of aspect 126 or 127, optionally as part of    a vector (eg, an expression vector).-   130. The nucleotide sequence of aspect 129, wherein the sequence is    a cDNA derived from a B-cell of the vertebrate from which the    antibody of aspect 126 is isolated, or is identical to such a cDNA.-   131. An isolated host cell (eg, a hybridoma or a CHO cell or a    HEK293 cell) comprising a nucleotide sequence according to aspect    129 or 130.-   132. A method of isolating an antibody that binds a predetermined    antigen, the method comprising    -   (a) providing a vertebrate (optionally a mammal; optionally a        mouse or rat according to any one of the above configurations,        examples, embodiments or aspects;    -   (b) immunising said vertebrate with said antigen;    -   (c) removing B lymphocytes from the vertebrate and selecting a B        lymphocyte expressing an antibody that binds to the antigen with        sub-nM affinity, wherein the antibody is according to aspect        126;    -   (d) optionally immortalising said selected B lymphocyte or        progeny thereof, optionally by producing hybridomas therefrom;        and    -   (e) isolating an antibody (eg, and IgG-type antibody) expressed        by the B lymphocyte.-   133. The method of aspect 132, comprising the step of isolating from    said B lymphocyte nucleic acid encoding said antibody that binds    said antigen; optionally exchanging the heavy chain constant region    nucleotide sequence of the antibody with a nucleotide sequence    encoding a human or humanised heavy chain constant region and    optionally affinity maturing the variable region of said antibody;    and optionally inserting said nucleic acid into an expression vector    and optionally a host.-   134. The method of aspect 132 or 133, further comprising making a    mutant or derivative of the antibody produced by the method of    aspect 132 or 133.

Inactivation by Inversion of Endogenous VDJ to Genome Desert Regions

-   135. A mouse or mouse cell comprising inverted endogenous heavy    chain gene segments (eg, VH, D and JH, such as the entire endogenous    heavy chain VDJ region) that are immediately 3′ of position    119753123, 119659458 or 120918606 on an endogenous mouse chromosome    12, wherein the mouse comprises a transgenic heavy chain locus    comprising a plurality of human VH gene segments, a plurality of    human D segments and a plurality of human JH segments operably    connected upstream of an endogenous constant region (eg, C mu) so    that the mouse or cell (optionally following differentiation into a    B-cell) is capable of expressing an antibody comprising a variable    region comprising sequences derived from the human gene segments.-   136. The mouse or cell of aspect 135, wherein the genome of the    mouse or cell is homozygous for said chromosome 12.-   137. A cassette for inversion and inactivation of endogenous    non-human vertebrate (eg, mouse or rat) antibody chain gene    segments, the segments being part of an antibody chain locus    sequence on a chromosome of a non-human vertebrate (eg, mouse or    rat) cell (eg, ES cell) wherein the sequence is flanked at its 3′    end by a site-specific recombination site (eg, lox, rox or frt), the    cassette comprising a nucleotide sequence encoding an expressible    label or selectable marker and a compatible site-specific    recombination site (eg, lox, rox or frt) flanked by a 5′ and a 3′    homology arm, wherein the homology arms correspond to or are    homologous to adjacent stretches of sequence in the cell genome on a    different chromosome or on said chromosome at least 10 mb away from    the endogenous gene segments.-   138. A cassette for inversion and inactivation of endogenous mouse    antibody heavy chain gene segments, the segments being part of a    heavy chain locus sequence on chromosome 12 of a mouse cell (eg, ES    cell) wherein the sequence is flanked at its 3′ end by a    site-specific recombination site (eg, lox, rox or frt), the cassette    comprising a nucleotide sequence encoding an expressible label or    selectable marker and a compatible site-specific recombination site    (eg, lox, rox or frt) flanked by a 5′ and a 3′ homology arm,    wherein (i) the 5′ homology arm is mouse chromosome 12 DNA from    coordinate 119753124 to coordinate 119757104 and the 3′ homology arm    is mouse chromosome 12 DNA from coordinate 119749288 to    119753123; (ii) the 5′ homology arm is mouse chromosome 12 DNA from    coordinate 119659459 to coordinate 119663126 and the 3′ homology arm    is mouse chromosome 12 DNA from coordinate 119656536 to 119659458;    or (iii) the 5′ homology arm is mouse chromosome 12 DNA from    coordinate 120918607 to coordinate 120921930 and the 3′ homology arm    is mouse chromosome 12 DNA from coordinate 120915475 to 120918606.-   139. A method of inactivating gene segments of an endogenous    antibody locus, the method comprising    -   (i) Providing a non-human vertebrate cell (eg, an ES cell, eg, a        mouse ES cell) whose genome comprises an antibody chain locus        comprising endogenous variable region gene segments;    -   (ii) Targeting a site-specific recombination site to flank the        3′ of the 3′-most of said endogenous gene segments;    -   (iii) Targeting a second site-specific recombination site at        least 10 mb away from said endogenous gene segments, the second        site being compatible with the first site inverted with respect        to the first site;    -   (iv) Expressing a recombinase compatible with said sites to        effect site-specific recombination between said sites, thereby        inverting and moving said gene segments away from said locus,        wherein the endogenous gene segments are inactivated; and    -   (v) Optionally developing the cell into a progeny cell or        vertebrate (eg, mouse or rat) whose genome is homozygous for the        inversion.-   140. A mouse or mouse cell whose genome comprises an inversion of a    chromosome 12, wherein the inversion comprises inverted endogenous    heavy chain gene segments (eg, VH, D and JH, such as the entire    endogenous heavy chain VDJ region); wherein the mouse comprises a    transgenic heavy chain locus comprising a plurality of human VH gene    segments, a plurality of human D segments and a plurality of human    JH segments operably connected upstream of an endogenous constant    region (eg, C mu) so that the mouse or cell (optionally following    differentiation into a B-cell) is capable of expressing an antibody    comprising a variable region comprising sequences derived from the    human gene segments; and wherein the inversion is (i) an inversion    of mouse chromosome 12 from coordinate 119753123 to coordinate    114666436; (ii) an inversion of mouse chromosome 12 from coordinate    119659458 to coordinate 114666436; or (iii) an inversion of mouse    chromosome 12 from coordinate 12091806 to coordinate 114666436.

Other aspects include:

A method for producing an antibody specific to a desired antigen themethod comprising immunizing a non-human mammal as disclosed herein withthe desired antigen and recovering the antibody or a cell producing theantibody.

A method for producing a fully humanised antibody comprising immunizinga non-human mammal as disclosed herein and then replacing the non-humanmammal constant region of an antibody specifically reactive with theantigen with a human constant region, suitably by engineering of thenucleic acid encoding the antibody.

A method, cell or mammal as disclosed herein wherein a human codingregion DNA sequence is in a functional arrangement with a non-humanmammal control sequence, such that transcription of the DNA iscontrolled by the non-human mammal control sequence. In one aspect thehuman coding region V, D or J region is in a functional arrangement witha mouse promoter sequence.

The invention also relates to a humanised antibody produced according toany methods disclosed herein and use of a humanised antibody so producedin medicine.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-8 show an iterative process for insertion of a series of humanBACs into a mouse Ig locus

FIGS. 9-18 show in more detail the process of FIGS. 1-8 for the IgH andkappa locus

FIGS. 19 and 20 show the principles behind antibody generation inchimaeric mice

FIG. 21 shows a possible insertion site for the human DNA in a mousechromosome

FIGS. 22-26 disclose an alternative iterative process for insertion of aseries of human BACs into a mouse Ig locus

FIGS. 27-29 illustrate a mechanism for inversion of the host VDJ region

FIG. 30 illustrates proof of principle for insertion of a plasmid usingan RMCE approach

FIG. 31 illustrates sequential RMCE—Integration into Landing Pad

FIG. 32 illustrates confirmation of Successful Insertion into LandingPad

FIG. 33 illustrates PCR Confirmation of 3′ End Curing

FIG. 34 illustrates insertion of BAC#1 and PCR Diagnostics

FIG. 35 illustrates JH and JK usage

FIG. 36 illustrates DH usage

FIG. 37 illustrates the distribution of CDR-H3 length in human VDJCμtranscripts from chimera mice

FIG. 38 illustrates the distribution of nucleotide numbers of deletionand insertion in IGH-VDI or IGK-VJ junctions

FIG. 39 illustrates Distribution of JH Usage Within Each VHs

FIG. 40 illustrates Distribution of DH Usage Within Each VHs

FIG. 41 illustrates Nucleotide Gain or Loss at VJ Joints Generates IGKVariants

FIG. 42 illustrates Hypermutaion in J Regions Generates IGK Variants

FIG. 43 illustrates Joint Diversity Produces Functional CDS

FIG. 44 illustrates a plot of identity of J_(H) gene segment use a5′-RACE Cμ-specific library generated from the splenic B lymphocytes oftransgenic mice according to the invention in which endogenous genesegment use has been inactivated by inversion

FIG. 45 illustrates the ratio of mouse V_(H) to human V_(H) usage asdetermined from antibody sequences from splenic B lymphocytes oftransgenic mice according to the invention in which endogenous genesegment use has been inactivated by inversion

FIG. 46 illustrates inversion strategy schematic

FIG. 47 illustrates targeting construct R57 for inversion

FIG. 48 illustrates sequence analysis from a Cμ-specific 5′-RACE libraryof splenic B lymphocytes of S1^(inv1) (one human IGH BAC (ie, multiplehuman VH, all functional human D and JH) with an inverted endogenous IGHlocus) mouse shows that practically all the transcripts came fromrearranged human V_(H)-D-J_(H) gene segments

FIG. 49 illustrates that the S1^(inv1) mouse shows a similar usage ofboth D and J_(H) gene segments to human

FIG. 50 illustrates that mouse V_(H) usage is further significantlyreduced following insertion of the 2^(nd) human BAC into the endogenousheavy chain locus

FIG. 51 illustrates a gel showing that normal class-switching (toIgG-type) was observed in transcripts from mice of the invention. Therearranged transcripts were detected using RT-PCR with human VH-specificand mouse Cγ-specific primers for amplification from peripheral bloodcells of immunized transgenic mice

FIG. 52 illustrates sequence analysis amplified fragments demonstratehypermutation occurred within the human variable regions of these IGγchains from mice of the invention

FIG. 53 illustrates Flow cytometric analysis showing normal B-cellcompartments in transgenic mice of the invention

FIGS. 54A-54D illustrate normal IgH isotypes in transgenic mice (H1)immunised with 100 μg Cholera Toxin B subunit. FIGS. 54E-54H illustratenormal IgH isotypes in transgenic mice (S1) immunised with 100 μgCholera Toxin B subunit.

FIG. 55A and FIG. 55B illustrate normal IgH isotypes and serum levelsare obtained in transgenic H1 and S1 animals, respectively, of theinvention following immunisation with antigens.

SEQUENCES

SEQ ID No 1 is a Rat switch sequence

SEQ ID No 2 is a landing pad targeting vector (long version)

SEQ ID No 3 is a landing pad targeting vector (shorter version)

SEQ ID No 4 is the mouse strain 129 switch

SEQ ID No 5 is the mouse strain C57 switch

SEQ ID No 6 is the 5′ homology arm of a landing pad

SEQ ID No 7 is oligo HV2-5

SEQ ID No 8 is oligo HV4-4

SEQ ID No 9 is oligo HV1-3

SEQ ID No 10 is oligo HV1-2

SEQ ID No 11 is oligo HV6-1

SEQ ID No 12 is oligo Cμ

SEQ ID No 13 is oligo KV1-9

SEQ ID No 14 is oligo KV1-8

SEQ ID No 15 is oligo KV1-6

SEQ ID No 16 is oligo KV1-5

SEQ ID No 17 is oligo Cκ

SEQ ID Nos 18-20 are rat switch sequences

SEQ ID No 21 is X₁X₂ T F G Q, where X₁X₂=PR, RT, or PW

SEQ ID No 22 is X₁X₂ T F G Q G T K V E I K R A D A, where X₁X₂=PR, RT,or PW;

SEQ ID No 23 is X₃X₄ T F G Q, where X₃X₄=PR or PW

SEQ ID No 24 is X₃X₄ T F G Q G T K V E I K R A D A, where X₃X₄=PR or PW

SEQ ID No 25 is Primer E1554

SEQ ID No 26 is Primer E1555

SEQ ID No 27 is Primer ELP1352_Cγ1

SEQ ID No 28 is Primer ELP1353_Cγ2b

SEQ ID No 29 is Primer ELP1354_Cγ2a

SEQ ID No 30 is Primer ELP1356VH4-4

SEQ ID No 31 is Primer ELP1357VH1-2,3

SEQ ID No 32 is Primer ELP1358VH6-1

SEQ ID No 33 is Primer mIgG1_(—)2 rev

SEQ ID No 34 is Primer mIgG2b rev

SEQ ID No 35 is Primer mIgG2a_(—)2 rev

SEQ ID No 36 is Primer mCH1 unirev

SEQ ID No 37 is Primer mCH1 unirev_(—)2

SEQ ID Nos 38-45 are CDRH3 sequences

SEQ ID Nos 46-50 is 3, 4, 5, 6 or more (up to 82) repeats of GGGCT

SEQ ID NOs 51-55 are heavy chain CDR1 sequences against CTB (cloned andreference)

SEQ ID NOs 56-60 are heavy chain CDR2 sequences against CTB (cloned andreference)

SEQ ID NOs 61-63 are heavy chain CDR3 sequences against CTB (cloned andreference)

SEQ ID NOs 64-68 are J Region sequences against CTB (cloned andreference)

DETAILED DESCRIPTION OF THE INVENTION

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims.

The use of the word “a” or an when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term or in the claims is usedto mean “and/or” unless explicitly indicated to refer to alternativesonly or the alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and “and/or.”Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term or combinations thereof as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As a source of antibody gene segment sequences, the skilled person willalso be aware of the following available databases and resources(including updates thereof) the contents of which are incorporatedherein by reference:

The Kabat Database (G. Johnson and T. T. Wu, 2002; World Wide Web (www)kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in 1966, theKabat database publishes aligned sequences of antibodies, T-cellreceptors, major histocompatibility complex (MHC) class I and IImolecules, and other proteins of immunological interest. A searchableinterface is provided by the SeqhuntII tool, and a range of utilities isavailable for sequence alignment, sequence subgroup classification, andthe generation of variability plots. See also Kabat, E. A., Wu, T. T.,Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteinsof Immunological Interest, 5th ed., NIH Publication No. 91-3242,Bethesda, Md., which is incorporated herein by reference, in particularwith reference to human gene segments for use in the present invention.

KabatMan (A. C. R. Martin, 2002; World Wide Web (www)bioinf.org.uk/abs/simkab.html). This is a web interface to make simplequeries to the Kabat sequence database.

IMGT (the International ImMunoGeneTics Information System®; M.-P.Lefranc, 2002; World Wide Web (www) imgt.cines.fr). IMGT is anintegrated information system that specializes in antibodies, T cellreceptors, and MHC molecules of all vertebrate species. It provides acommon portal to standardized data that include nucleotide and proteinsequences, oligonucleotide primers, gene maps, genetic polymorphisms,specificities, and two-dimensional (2D) and three-dimensional (3D)structures. IMGT includes three sequence databases (IMGT/LIGM-DB,IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3Dstructure database (IMGT/3Dstructure-DB), and a range of web resources(“IMGT Marie-Paule page”) and interactive tools.

V-BASE (I. M. Tomlinson, 2002; World Wide Web (www)mrc-cpe.cam.ac.uk/vbase). V-BASE is a comprehensive directory of allhuman antibody germline variable region sequences compiled from morethan one thousand published sequences. It includes a version of thealignment software DNAPLOT (developed by Hans-Helmar Althaus and WernerMüller) that allows the assignment of rearranged antibody V genes totheir closest germline gene segments.

Antibodies—Structure and Sequence (A. C. R. Martin, 2002; World Wide Web(www) bioinf.org.uk/abs). This page summarizes useful information onantibody structure and sequence. It provides a query interface to theKabat antibody sequence data, general information on antibodies, crystalstructures, and links to other antibody-related information. It alsodistributes an automated summary of all antibody structures deposited inthe Protein Databank (PDB). Of particular interest is a thoroughdescription and comparison of the various numbering schemes for antibodyvariable regions.

AAAAA (A Ho's Amazing Atlas of Antibody Anatomy; A. Honegger, 2001;World Wide Web (www) unizh.ch/-antibody). This resource includes toolsfor structural analysis, modeling, and engineering. It adopts a unifyingscheme for comprehensive structural alignment of antibody andT-cell-receptor sequences, and includes Excel macros for antibodyanalysis and graphical representation.

WAM (Web Antibody Modeling; N. Whitelegg and A. R. Rees, 2001; WorldWide Web (www) antibody.bath.ac.uk). Hosted by the Centre for ProteinAnalysis and Design at the University of Bath, United Kingdom. Based onthe AbM package (formerly marketed by Oxford Molecular) to construct 3Dmodels of antibody Fv sequences using a combination of establishedtheoretical methods, this site also includes the latest antibodystructural information.

Mike's Immunoglobulin Structure/Function Page (M. R. Clark, 2001; WorldWide Web (www) path.cam.ac.uk/˜mrc7/mikeimages.html) These pages provideeducational materials on immunoglobulin structure and function, and areillustrated by many colour images, models, and animations. Additionalinformation is available on antibody humanization and Mike Clark'sTherapeutic Antibody Human Homology Project, which aims to correlateclinical efficacy and anti-immunoglobulin responses with variable regionsequences of therapeutic antibodies.

The Antibody Resource Page (The Antibody Resource Page, 2000; World WideWeb (www) antibodyresource.com). This site describes itself as the“complete guide to antibody research and suppliers.” Links to amino acidsequencing tools, nucleotide antibody sequencing tools, andhybridoma/cell-culture databases are provided.

Humanization by Design (J. Saldanha, 2000; World Wide Web (www)people.cryst.bbk.ac.uk/˜ubcg07s). This resource provides an overview onantibody humanization technology. The most useful feature is asearchable database (by sequence and text) of more than 40 publishedhumanized antibodies including information on design issues, frameworkchoice, framework back-mutations, and binding affinity of the humanizedconstructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo,Methods in Molecular Biology™, Human Press. Also at World Wide Web (www)blogsua.com/pdf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention.

EXAMPLES Example 1 BAC Recombineering

Overall Strategy:

A mouse model of the invention can be achieved by inserting ˜960 kb ofthe human heavy chain locus containing all the V, D and J-regionsupstream of the mouse constant region and 473 kb of the human kapparegion upstream of the mouse constant region. Alternatively, or intandem, the human lambda region is inserted upstream of the mouseconstant region. This insertion is achieved by gene targeting in EScells using techniques well known in the art.

High fidelity insertion of intact V-D-J regions into each locus in theirnative (wild-type) configuration is suitably achieved by insertion ofhuman bacterial artificial chromosomes (BACs) into the locus. Suitablythe BACs are trimmed so that in the final locus no sequence isduplicated or lost compared to the original. Such trimming can becarried out by recombineering.

The relevant human BACs, suitably trimmed covering these loci are onaverage 90 kb in size.

In one approach the full complement of human D and J-elements as well asseven or eight human V-regions are covered by the first BACs to beinserted in the experimental insertion scheme described below. The firstBACs to be inserted in the IgH and IgK loci may contain the followingV-regions. IgH:V6-1, VII-1-1, V1-2, VIII-2-1, V1-3, V4-4, V2-5 and IgK:V4-1, V5-2, V7-3, V2-4, V1-5, V1-6, V3-7, V1-8.

Suitably the performance of each locus is assessed after the first BACinsertion using chimaeric mice and also after each subsequent BACaddition. See below for detailed description of this performance test.

Nine additional BAC insertions will be required for the IgH locus andfive for IgK to provide the full complement of human V-regions coveringall 0.96 Mb and 0.473 Mb of the IgH and IgK loci, respectively.

Not all BACs retain their wild-type configuration when inserted into theES cell genome. Thus, high density genomic arrays were deployed toscreen ES cells to identify those with intact BAC insertions (Barrett,M. T., Scheffer, A., Ben-Dor, A., Sampas, N., Lipson, D., Kincaid, R.,Tsang, P., Curry, B., Baird, K., Meltzer, P. S., et al. (2004).Comparative genomic hybridization using oligonucleotide microarrays andtotal genomic DNA. Proceedings of the National Academy of Sciences ofthe United States of America 101, 17765-17770.). This screen alsoenables one to identify and select against ES clones in which the EScell genome is compromised and thus not able to populate the germ lineof chimeric animals. Other suitable genomic tools to facilitate thisassessment include sequencing and PCR verification.

Thus in one aspect the correct BAC structure is confirmed before movingto the next step.

It is implicit from the description above that in order to completelyengineer the loci with 90 kb BACs, it is necessary to perform a minimumof 10 targeting steps for IgH and 5 steps for the IgK. Mice with an IgLlocus can be generated in a similar manner to the IgK locus. Additionalsteps are required to remove the selection markers required to supportgene targeting. Since these manipulations are being performed in EScells in a step-wise manner, in one aspect germ line transmissioncapacity is retained throughout this process.

Maintaining the performance of the ES cell clones through multiplerounds of manipulation without the need to test the germ line potentialof the ES cell line at every step may be important in the presentinvention. The cell lines currently in use for the KOMP and EUCOMMglobal knockout projects have been modified twice prior to their use forthis project and their germ line transmission rates are unchanged fromthe parental cells (these lines are publicly available, see World WideWeb (www) komp.org and World Wide Web (www) eucomm.org). This cell line,called JM8, can generate 100% ES cell-derived mice under publishedculture conditions (Pettitt, S. J., Liang, Q., Rairdan, X. Y., Moran, J.L., Prosser, H. M., Beier, D. R., Lloyd, K. C., Bradley, A., andSkarnes, W. C. (2009). Agouti C57BL/6N embryonic stem cells for mousegenetic resources. Nature Methods.). These cells have demonstratedability to reproducibly contribute to somatic and germ line tissue ofchimaeric animals using standard mouse ES cell culture conditions. Thiscapability can be found with cells cultured on a standard feeder cellline (SNL) and even feeder-free, grown only on gelatine-coated tissueculture plates. One particular sub-line, JM8A3, maintained the abilityto populate the germ line of chimeras after several serial rounds ofsub-cloning. Extensive genetic manipulation via, for example, homologousrecombination—as would be the case in the present invention—cannotcompromise the pluripotency of the cells. The ability to generatechimeras with such high percentage of ES cell-derived tissue has otheradvantages. First, high levels of chimerism correlates with germ linetransmission potential and provide a surrogate assay for germ linetransmission while only taking 5 to 6 weeks. Second, since these miceare 100% ES cell derived the engineered loci can be directly tested,removing the delay caused by breeding. Testing the integrity of the newIg loci is possible in the chimera since the host embryo will be derivedfrom animals that are mutant for the RAG-1 gene as described in the nextsection.

Another cell line that may be used is an HPRT-ve cell line, such asAB2.1, as disclosed in Ramirez-Solis R, Liu P and Bradley A, “Chromosomeengineering in mice,” Nature, 1995; 378; 6558; 720-4.

RAG-1 Complementation:

While many clones will generate 100% ES derived mice some will not.Thus, at every step mice are generated in a RAG-1-deficient background.This provides mice with 100% ES-derived B- and T-cells which can be useddirectly for immunization and antibody production. Cells having a RAG-2deficient background, or a combined RAG-1/RAG-2 deficient background maybe used, or equivalent mutations in which mice produce only EScell-derived B cells and/or T cells.

In order that only the human-mouse IgH or IgK loci are active in thesemice, the human-mouse IgH and IgK loci can be engineered in a cell linein which one allele of the IgH or IgK locus has already beeninactivated. Alternatively the inactivation of the host Ig locus, suchas the IgH or IgK locus, can be carried out after insertion.

Mouse strains that have the RAG-1 gene mutated are immunodeficient asthey have no mature B- or T-lymphocytes (U.S. Pat. No. 5,859,307). T-and B-lymphocytes only differentiate if proper V(D)J recombinationoccurs. Since RAG-1 is an enzyme that is crucial for this recombination,mice lacking RAG-1 are immunodeficient. If host embryos are geneticallyRAG-1 homozygous mutant, a chimera produced by injecting such an embryowill not be able to produce antibodies if the animal's lymphoid tissuesare derived from the host embryo. However, JM8 cells and AB2.1 cells,for example, generally contribute in excess of 80% of the somatictissues of the chimeric animal and would therefore usually populate thelymphoid tissue. JM8 cells have wild-type RAG-1 activity and thereforeantibodies produced in the chimeric animal would be encoded by theengineered JM8 ES cell genome only. Therefore, the chimeric animal canbe challenged with an antigen by immunization and subsequently produceantibodies to that antigen. This allows one skilled in the art to testthe performance of the engineered human/mouse IgH and IgK loci asdescribed in the present invention. See FIGS. 19 and 20.

One skilled in the art would use the chimeric animal as described todetermine the extent of antibody diversity (see e.g. Harlow, E. & Lane,D. 1998, 5^(th) edition, Antibodies: A Laboratory Manual, Cold SpringHarbor Lab. Press, Plainview, N.Y.). For example, the existence in thechimeric animal's serum of certain antibody epitopes could beascertained by binding to specific anti-idiotype antiserum, for example,in an ELISA assay. One skilled in the art could also sequence thegenomes of B-cell clones derived from the chimeric animal and comparesaid sequence to wild-type sequence to ascertain the level ofhypermutation, such hypermutation indicative of normal antibodymaturation.

One skilled in the art would also use said chimeric animal to examineantibody function wherein said antibodies are encoded from theengineered Ig loci (see e.g. Harlow, E. & Lane, D. 1998, 5^(th) edition,Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press,Plainview, N.Y.). For example, antisera could be tested for binding anantigen, said antigen used to immunize the chimeric animal. Such ameasurement could be made by an ELISA assay. Alternatively, one skilledin the art could test for neutralization of the antigen by addition ofthe antisera collected from the appropriately immunized chimeric animal.

It is well known to those skilled in the art that positive outcomes forany of these tests demonstrate the ability of the engineered Ig loci,the subject of the instant invention, to encode antibodies with humanvariable regions and mouse constant regions, said antibodies capable offunctioning in the manner of wild-type antibodies.

Experimental Techniques:

Recombineering for the production of vectors for use in homologousrecombination in ES cells is disclosed in, for example, WO9929837 andWO0104288, and the techniques are well known in the art. In one aspectthe recombineering of the human DNA takes place using BACs as a sourceof said human DNA. Human BAC DNA will be isolated using QIAGEN®, BACpurification kit. The backbone of each human BAC will be modified usingrecombineering to the exact same or similar configuration as the BACalready inserted into the mouse IgH region. The genomic insert of eachhuman BAC will be trimmed using recombineering so that once the BACs areinserted, a seamless contiguous part of the human V(D)J genomic regionwill form at the mouse IgH or IgK locus. BAC DNA transfection byelectroporation and genotyping will be performed accordingly to standardprotocols (Prosser, K M., Rzadzinska, A. K., Steel, K. P., and Bradley,A. (2008). “Mosaic complementation demonstrates a regulatory role formyosin Vila in actin dynamics of stereocilia.” Molecular and CellularBiology 28, 1702-1712; Ramirez-Solis, R., Davis, A. C., and Bradley, A.(1993). “Gene targeting in embryonic stem cells.” Methods in Enzymology225, 855-878.). Recombineering will be performed using the proceduresand reagents developed by Pentao Liu and Don Court's laboratories (Chan,W., Costantino, N., Li, R., Lee, S. C., Su, Q., Melvin, D., Court, D.L., and Liu, P. (2007). “A recombineering based approach forhigh-throughput conditional knockout targeting vector construction.”Nucleic Acids Research 35, e64).

These and other techniques for gene targeting and recombination ofBAC-derived chromosomal fragments into a non-human mammal genome, suchas a mouse are well-known in the art and are disclosed in, for example,in World Wide Web (www) eucomm.org/information/targeting and World WideWeb (www)eucomm.org/information/publications.

Cell culture of C57BL/6N-derived cell lines, such as the JM8 male EScells will follow standard techniques. The JM8 ES cells have been shownto be competent in extensively contributing to somatic tissues and tothe germline, and are being used for large mouse mutagenesis programs atthe Sanger Institute such as EUCOMM and KOMP (Pettitt, S. J., Liang, Q.,Rairdan, X. Y., Moran, J. L., Prosser, H. M., Beier, D. R., Lloyd, K.C., Bradley, A., and Skarnes, W. C. (2009). “Agouti C57BL/6N embryonicstem cells for mouse genetic resources.” Nature Methods.). JM8 ES cells(1.0×10⁷) will be electroporated (500 μF, 230V; Bio-Rad®) with 10 μgI-Scel linearized human BAC DNA. The transfectants will be selected witheither Puromycin (3 μg/ml) or G418 (150 μg/ml). The selection will begineither 24 hours (with G418) or 48 hours (with Puromycin) postelectroporation and proceed for 5 days. 10 μg linearized human BAC DNAcan yield up to 500 Puromycin or G418 resistant ES cell colonies. Theantibiotic resistant ES cell colonies will be picked into 96-well cellculture plates for genotyping to identify the targeted clones.

Once targeted mouse ES cell clones are identified, they will be analyzedby array Comparative Genomic Hybridization (CGH) for total genomeintegrity (Chung, Y. J., Jonkers, J., Kitson, H., Fiegler, H., Humphray,S., Scott, C., Hunt, S., Yu, Y., Nishijima, I., Velds, A., et al.(2004). “A whole-genome mouse BAC microarray with 1-Mb resolution foranalysis of DNA copy number changes by array comparative genomichybridization.” Genome research 14, 188-196. and Liang, Q., Conte, N.,Skarnes, W. C., and Bradley, A. (2008). “Extensive genomic copy numbervariation in embryonic stem cells.” Proceedings of the National Academyof Sciences of the United States of America 105, 17453-17456.). ES cellsthat have abnormal genomes do not contribute to the germline of thechimeric mice efficiently. BAC integrity will be examined byPCR-amplifying each known functional V gene in the BAC. For example, inone approach the first human BAC chosen for the IgH locus has 6functional V genes. To confirm the integrity of this BAC for thepresence of these 6 IGH V genes, at least 14 pairs of PCR primers willbe designed and used to PCR-amplify genomic DNA from the targeted EScells. The human wild-type size and sequence of these fragments willensure that the inserted BAC has not been rearranged.

More detailed CGH will also confirm the integrity of the inserted BACs.For example, one skilled in the art could use an oligo aCGH platform,which is developed by Agilent Technologies, Inc. This platform not onlyenables one to study genome-wide DNA copy number variation at highresolution (Barrett, M. T., Scheffer, A., Ben-Dor, A., Sampas, N.,Lipson, D., Kincaid, R., Tsang, P., Curry, B., Baird, K., Meltzer, P.S., et al. (2004). “Comparative genomic hybridization usingoligonucleotide microarrays and total genomic DNA.” Proceedings of theNational Academy of Sciences of the United States of America 101,17765-17770.), but permit examination of a specific genome region usingcustom designed arrays. Comparing the traditional aCGH techniques whichrely on cDNA probes or whole BAC probes, the 60-mer oligonucleotidesprobes can ensure specific hybridization and high sensitivity andprecision that is needed in order to detect the engineered chromosomealterations that were made. For example, oligos designed to hybridize atregular intervals along the entire length of the inserted BAC woulddetect even quite short deletions, insertions or other rearrangements.Also, this platform provides the greatest flexibility for customizedmicroarray designs. The targeted ES cell genomic DNA and normal humanindividual genomic DNA will be labelled separately with dyes andhybridized to the array. Arrays slides will be scanned using an AglientTechnologies DNA microarray scanner. Reciprocal fluorescence intensitiesof dye Cγ5 and dye Cγ3 on each array image and the log 2 ratio valueswill be extracted by using Bluefuse software (Bluegnome). Spots withinconsistent fluorescence patterns (“confidence”<0.29 or “quality”=0)will be excluded before normalizing all log 2 ratio values. Within anexperiment, Log 2 ratio between −0.29 and +0.29 for the signal from anyoligo probe are regarded as no copy number change. The log 2 ratiothreshold for “Duplication” is usually >0.29999, and for deletion is<0.29999.

Once the first human BAC is inserted into the mouse IgH locus andconfirmed to be in its intact, native configuration, the FRT-flanked BACbackbone will be excised by using Flp site-specific recombinase. Ifregular Flp-catalyzed FRT recombination is not high enough, one can useFlo, an improved version of Flpo recombinase which in certain tests is3-4 times more efficient than the original Flp in ES cells. After theBAC backbone is excised, ES cells will become sensitive to Puromycin (orG418) and resistant to FIAU (for loss of the TK cassette). The excisionevents will be further characterized by PCR amplification of thejunction fragment using human genomic DNA primers. These FRT-flanked BACbackbone-free ES cells will be used for the next round of human BACinsertion and for blastocyst injection.

Targeting of the genome of an ES cell to produce a transgenic mouse maybe carried out using a protocol as explained by reference to theattached FIGS. 1-18.

FIG. 1 illustrates three basic backbone vectors; an initiating cassetteand 2 large insert vectors 1 and 2 respectively. The initiating cassettecomprises sequences homologous to the desired site of insertion into themouse genome, those sites flanking a selectable marker and stufferprimer sequence for PCR based genotyping to confirm correct insertion ofBACs. The Stuffer-primer sequence provides the basis for genotyping eachBAC addition step. This sequence is considered to provide a robust wellvalidated sequence template for PCR primer and may be located at theIScel site, ideally ˜1 kb from the BAC insert.

The large insert vectors comprise human DNA on plasmids with selectablemarkers and a unique restriction site for linearisation of the plasmidto aid in homologous recombination into the genome of the ES cell.

FIG. 2 illustrates insertion of an initiating cassette into the mousegenome by Homologous recombination between the mouse J4 and C alphaexons. Puromycin selection allows identification of ES cells withinsertion of the cassette. pu(Delta)tk is a bifunctional fusion proteinbetween puromycin N-acetyltransferase (Puro) and a truncated version ofherpes simplex virus type 1 thymidine kinase (DeltaTk). Murine embryonicstem (ES) cells transfected with pu(Delta)tk become resistant topuromycin and sensitive to1-(-2-deoxy-2-fluoro-1-beta-D-arabino-furanosyl)-5-iodouracil (FIAU).Unlike other HSV1 tk transgenes, puDeltatk is readily transmittedthrough the male germ line. Thus pu(Delta)tk is a convenientpositive/negative selectable marker that can be widely used in many EScell applications.

FIG. 3 illustrates targeting of the large insert vector 1 to the mouseES cell genome. Linearisation of the vector is made at the same positionas the stuffer primer sequence which allows for a gap repair genotypingstrategy, well known in the art—see Zheng et al NAR 1999, Vol 27, 11,2354-2360. In essence, random insertion of the targeting vector into thegenome will not ‘repair’ the gap whereas a homologous recombinationevent will repair the gap. Juxtaposition of appropriate PCR primersequences allows colonies to be screened individually for a positive PCRfragment indicating proper insertion. Positive selection using G418allows for identification of mouse ES cells containing the neo selectionmarker. PCR verification can be made of all critical V, D and J regions.Array comparative genomic hybridization can be used to validate the BACstructure.

FIG. 4 illustrates the puro-delta-tk cassette and the BAC plasmidbackbone is deleted using Flpe and select in FIAU. Since Flpe worksinefficiently in mouse ES cells (5% deletion with transient Flpeexpression), it is expected that in most cases, the recombination occursbetween the two FRT sites flanking the BAC backbone. Flpo can also betested to find out the recombination efficiency between two FRT sitesthat are 10 kb away.

Given that the FRT deletion step is selectable it is possible to poolFIAU resistant clones and proceed immediately to the next step inparallel with clonal analysis. Alternatively it may be desirable to showby short range PCR that the human sequences are now adjacent to those ofthe mouse as shown (Hu-primer 1 and Mo-primer)

At this stage a 200 kb human locus will have been inserted,

FIG. 5 illustrates a second large insert vector is targeted into the EScell chromosome. The human BAC is targeted to the mouse IgH locus usingthe same initiation cassette insertion followed by IScel BAClinearization, BAC targeting to the initiation cassette and gap-repairgenotyping strategy. Verification of the BAC insertion is carried out asbefore.

FIG. 6 illustrates the FRTY flanked BAC backbone of large insert vector2 and the neo marker are deleted via Flpo. Note that this is notselectable, thus it will be necessary for clonal analysis at this point.This will enable confirmation of the juxtaposition of the human 2 insertwith human 1 and other validation efforts.

At this stage a ˜200 kb human locus will have been inserted.

FIG. 7 illustrates the next large insert vector targeted to the mouseIgH locus. The pu-delta TK cassette is then removed, as for FIG. 4. Theprocess can be repeated to incorporate other BACs.

FIG. 8 illustrates the final predicted ES cell construct.

FIGS. 9-18 provide a further level of detail of this process.

Example 2 Site-Specific Recombination

In a further method of the invention site specific recombination canalso be employed. Site-specific recombination (SSR) has been widely usedin the last 20-years for the integration of transgenes into definedchromosomal loci. SSR involves recombination between homologous DNAsequences.

The first generation of SSR-based chromosomal targeting involvedrecombination between (i) a single recombination target site (RT) suchas loxP or FRT in a transfected plasmid with (ii) a chromosomal RT siteprovided by a previous integration. A major problem with this approachis that insertion events are rare since excision is always moreefficient than insertion. A second generation of SSR called RMCE(recombinase-mediated cassette exchange) was introduced by Schlake andBode in 1994 (Schlake, T.; J. Bode (1994). “Use of mutatedFLP-recognition-target-(FRT-) sites for the exchange of expressioncassettes at defined chromosomal loci”. Biochemistry 33: 12746-12751).Their method is based on using two heterospecific and incompatible RTsin the transfected plasmid which can recombine with compatible RT siteson the chromosome resulting in the swap of one piece of DNA foranother—or a cassette exchange. This approach has been successfullyexploited in a variety of efficient chromosomal targeting, includingintegration of BAC inserts of greater than 50 kb (Wallace, H. A. C. etal. (2007). “Manipulating the mouse genome to engineering precisefunctional syntenic replacements with human sequence”. Cell 128:197-209; Prosser, H. M. et al. (2008). “Mosaic complementationdemonstrates a regulatory role for myosin Vila in actin dynamics ofStereocilia”. Mol. Cell. Biol. 28: 1702-12).

The largest insert size of a BAC is about 300-kb and therefore thisplaces an upper limit on cassette size for RMCE.

In the present invention a new SSR-based technique called sequentialRMCE (SRMCE) was used, which allows continuous insertion of BAC insertsinto the same locus.

The method comprises the steps of

-   -   1 insertion of DNA forming an initiation cassette (also called a        landing pad herein) into the genome of a cell;    -   2 insertion of a first DNA fragment into the insertion site, the        first DNA fragment comprising a first portion of a human DNA and        a first vector portion containing a first selectable marker or        generating a selectable marker upon insertion;    -   3 removal of part of the vector DNA;    -   4 insertion of a second DNA fragment into the vector portion of        the first DNA fragment, the second DNA fragment containing a        second portion of human DNA and a second vector portion, the        second vector portion containing a second selectable marker, or        generating a second selectable marker upon insertion;    -   5 removal of any vector DNA to allow the first and second human        DNA fragments to form a contiguous sequence; and    -   6 iteration of the steps of insertion of a part of the human        V(D)J DNA and vector DNA removal, as necessary, to produce a        cell with all or part of the human VDJ or VJ region sufficient        to be capable of generating a chimaeric antibody in conjunction        with a host constant region,        wherein the insertion of at least one DNA fragment uses site        specific recombination.

In one specific aspect the approach utilizes three heterospecific andincompatible loxP sites. The method is comprised of the steps asfollows, and illustrated in FIGS. 22-26:

-   -   1. Targeting a landing pad into the defined locus. An entry        vector containing an HPRT mini-gene flanked by inverted piggyBac        (PB) ITRs is targeted into defined region (for example: a region        between IGHJ and Eμ or IGKJ and EK or IGLC1 and EA3-1) to serve        as a landing pad for BAC targeting. The HPRT mini-gene is        comprised of two synthetic exons and associated intron. The 5′        HPRT exon is flanked by two heterospecific and incompatible loxP        sites (one wild-type and the other a mutated site, lox5171) in        inverted orientation to each other (FIG. 22). These two loxP        sites provide recombination sites for the BAC insertion through        RMCE.

-   2. Insertion of the 1^(st) modified BAC into the targeted landing    pad. The 1^(st) BAC has a length of DNA to be inserted into the    genome flanked by engineered modifications. The 5′ modification    (loxP-neo gene-lox2272-PGK promoter-PB 5′LTR) and 3′ modification    (PB3′LTR-puroΔTK gene-lox5171) is depicted in FIG. 23 along with the    relative orientations of the lox sites and PB LTRs. With transient    CRE expression from a co-electroporated vector, the DNA sequence    would be inserted into the defined locus through RMCE. The cells in    which a correct insertion has occurred can be selected as    follows: (i) Puromycin-resistance (the puroΔTK gene has acquired a    promoter—“PGK”—from the landing pad), (ii) 6TG-resistance (the HPRT    mini-gene has been disrupted), and (iii) G418-resistance (selects    for any insertion via the 5′ region PGK-neo arrangement). Any    combination of these selection regimes can be used. G418- and    6TG-resistance select for correct events on the 5′ end while    puro-resistance selects for correct events on the 3′ end.    -   3. Curing (removing) the 3′ modification of the 1^(st)        insertion. A properly inserted 1^(st) BAC results the 3′ end        having a puroΔTK gene flanked by inverted PB LTRs (FIG.        24)—essentially a proper transposon structure. This transposon        can then be removed by the transient expression of the piggyBac        transposase (from an electroporated vector). Cells with the        correct excision event can be selected by FIAU resistance—ie, no        thymidine kinase activity from the puroΔTK gene. This completely        removes the 3′ modification leaving no trace nucleotides.    -   4. Insertion of a 2^(nd) modified BAC into the 5′ end of 1^(st)        insertion. The 2^(nd) BAC has a length of DNA to be inserted        into the genome (usually intended to be contiguous with the DNA        inserted with the 1^(st) BAC) flanked by engineered        modifications. The 5′ modification (loxP-HPRT mini gene 5′        portion-lox5171-PGK promoter-PB5′LTR) and 3′ modification        (PB3′LTR-puroΔTK-lox2272) is depicted in FIG. 25 along with the        relative orientations of the lox sites and PB LTRs. With        transient CRE expression from a co-electroporated vector, the        DNA sequence would be inserted into the defined locus through        RMCE. The cells in which a correct insertion has occurred can be        selected as follows: (i) HAT-resistance (the HPRT mini-gene is        reconstituted by a correct insertion event, ie: the 5′ and 3′        exon structures are brought together), and (ii)        puromycin-resistance (puroΔTK gene has acquired a        promoter—“PGK”—from the landing pad).    -   5. Curing (removing) the 3′ modification of the 2^(nd)        insertion. A properly inserted 2^(nd) BAC results the 3′ end        having a puroΔTK gene flanked by inverted PB LTRs (FIG.        26)—essentially a proper transposon structure, exactly analogous        to the consequence of a successful 1^(st) BAC insertion. And        therefore this transposon can likewise be removed by the        transient expression of the piggyBac transposase (from an        electroporated vector). Cells with the correct excision event        can be selected by FIAU resistance—ie, no thymidine kinase        activity from the puroΔTK gene. This completely removes the 3′        modification leaving no trace nucleotides.    -   6. After curing of the 3′ modification of the 2^(nd) BAC        insertion, the landing pad becomes identical to the original.        This entire process, steps 2 through 5, can be repeated multiple        times to build up a large insertion into the genome. When        complete, there are no residual nucleotides remaining other than        the desired insertion.

With the insertion of an odd number of BACs into the Ig loci, theendogenous VDJ or VJ sequences can be inactivated through an inversionvia chromosomal engineering as follows (see FIGS. 27-29):

-   -   1. Targeting a “flip-over” cassette into a 5′ region 10 to 40        megabases away from the endogenous VDJ or VJ. The flip-over        vector (PB3′LTR-PGK promoter-HPRT mini gene 5′        portion-loxP-puroΔTK-CAGGS promoter-PB3′LTR) is depicted in FIG.        27 along with the relative orientations of the lox sites and PB        LTRs.    -   2. Transient CRE expression will result in recombination between        the loxP site in the “flip-over” cassette and the loxP site in        the 5′ modification. This 5′ modification is as described in        Steps 2 and 3 above—essentially the modification resulting from        insertion of an odd number of BACs, after the 3′ modification        has been cured. The loxP sites are inverted relative to one        another and therefore the described recombination event results        in an inversion as depicted in FIG. 28. Cells with the correct        inversion will be HAT-resistance since the HPRT mini-gene is        reconstituted by a correct inversion.    -   3. A correct inversion also leaves two transposon structures        flanking the “flip-over” cassette and the 5′ modification. Both        can be excised with transient piggyBAC transposase expression,        leaving no remnant of either modification (FIG. 29). Cells with        the correct excisions can be selected as follows: (i)        6TG-resistance (the HPRT mini-gene is deleted) and (ii)        FIAU-resistance (the puroΔTK gene is deleted). An inversion as        described in the Ig loci would move the endogenous IGH-VDJ or        IGK-VJ region away from the Eμ or EK enhancer region,        respectively, and lead to inactivation of the endogenous IGH-VDJ        or IGK-VJ regions.

The methods of insertion of the invention suitably provide one or moreof:

-   -   Selection at both 5′ and 3′ ends of the inserted DNA fragment;    -   Efficient curing of the 3′ modification, preferably by        transposase mediated DNA excision;    -   Inactivation of endogenous IGH or IGK activity through an        inversion; and    -   Excision of modifications, leaving no nucleotide traces        remaining in the chromosome.

Example 3 Insertion of a Test Vector into the Genome at a DefinedLocation

Proof of concept of the approach is disclosed in FIG. 30. In FIG. 30 alanding pad as shown in FIG. 22 was inserted into the genome of a mouseby homologous recombination, followed by insertion of the R21 plasmidinto that landing pad via cre-mediated site specific recombination. Theinsertion event generated a number of general insertion events, 360 G418resistant colonies, of which ˜220 were inserted into the desired locus,as demonstrated by disruption of the HRPT minilocus.

The R21 vector mimicks the 1^(st) BAC insertion vector at the 5′ and 3′ends, including all selection elements and recombinase target sites. Inplace of BAC sequences, there is a small ‘stuffer’ sequence. This vectorwill both test all the principals designed in the invention and alloweasy testing of the results in that PCR across the stuffer is feasibleand therefore allows both ends of the insertion to be easily tested. R21was co-electroporated with a cre-expressing vector into the ES cellsharbouring the landing pad in the IGH locus. Four sets of transformedcells were transfected in parallel and then placed under differentselection regimes as indicated in FIG. 30. G418 selection (neo geneexpression) resulted in the largest number of colonies due to therebeing no requirement for specific landing-pad integration. Anyintegration of R21 into the genome will provide neo expression leadingto G418-resistance. Puro selection resulted in a similar colony numberto Puro+6TG or G418+6TG, suggesting that the stringency of Puroselection is due to the PuroΔTK lacking a promoter in the vector. Puroexpression is only acquired when an integration occurs near a promoterelement—in this design most likely specifically in the landing pad.These conclusions are supported by the results from junction PCR whichis shown in FIG. 31.

The next step in the invention is to ‘cure’ the 3′ end of the integratedBAC vector, leaving a seamless transition between the insertion and theflanking genome. This curing was demonstrated by expanding an individualclone from above (R21 inserted into the landing pad) and expressingpiggyBac recombinase in this clone via transfection of an expressingplasmid. FIAU was used to select colonies in which the 3′ modificationwas excised—ie, through loss of the ‘PGK-puroΔTK’ element between thepiggyBac terminal repeats. Fifty such clones resulted from atransfection of 10⁶ cells; of these six were tested for the expectedgenomic structure. Successful curing resulted in positive PCR betweenthe primer set labelled “3” in FIG. 32. Of the 6 clones, 4 had correctexcisions, 1 clone remained in the original configuration and 1 otherhad a deletion.

These data demonstrate iterative insertion of DNA into a landing pad ata defined genomic locus using the approaches outlined above.

Example 4 Insertion of Large Parts of the Human IG Loci into DefinedPositions in the Mouse Genome

Example 3 demonstrated that the design of the claimed invention wascapable of providing for the insertion of a test vector into the genomeat a defined location, in this case the R21 vector into the mouse IGHlocus. The use of the appropriate selection media and the expression ofcre-recombinase resulted in a genomic alteration with the predictedstructure.

The same design elements described in this invention were built into the5′ and 3′ ends of a BAC insert. Said insert comprised human sequencesfrom the IGH locus and was approximately 166-kb. This engineered BAC waselectroporated along with a cre-expressing plasmid DNA into mouse EScells harbouring the landing pad at the mouse IGH locus. The transfectedcell population was grown in puro-containing media to select forappropriate insertion events.

Seven resulting clones were isolated and further analysed. The expectedrecombination event and resulting structure are depicted in FIG. 33.Based upon data from the R21 experiment outlined in Example 3, astringent selection for correct clones was expected when the transfectedpopulation was selected in puro-containing media. This is because thepuro-coding region requires a promoter element and this ispreferentially supplied by the landing pad after recombination.Accordingly, the majority of the 7 isolated clones had insertedcorrectly into the genome at the landing pad as determined by thediagnostic PCR. The primers for diagnosing a correct insertion aredepicted in FIG. 33. Correct junctions are present in the genome if a610-bp fragment is amplified between primers ‘A’ and ‘X’ and a 478-bpfragment is amplified between primers ‘Y’ and ‘B’ (FIGS. 33 and 34).Note that there are amplified fragments between ‘A’ and ‘1’ primers and‘2’ and ‘B’ primers indicating the presence of parental genome (that is,the landing pad alone). These result from parental cells presentinternally in the cell colonies under puro-selection that escape theselection due to the geometry of a colony. After passaging the colonythrough puro-containing media, these parental junction fragmentsdisappear indicating that the parental cells are removed from thepopulation. In addition, all the clones were shown to be resistant to6-TG as expected if the HPRT gene is inactivated by the correctinsertion event.

These data indicate that the disclosed strategy for inserting largeparts of the human IG loci into defined positions in the mouse genomewill enable the construction of a mouse with a plurality of the variableregions of human IG regions upstream of the mouse constant regions asdescribed.

Example 5 Inserted Loci are Functional in Terms of Gene Rearrangement,Junctional Diversity as Well as Expression

Bacterial artificial chromosomes (BACs) were created, wherein the BACshad inserts of human Ig gene segments (human V, D and/or J genesegments). Using methods described herein, landing pads were used in amethod to construct chimaeric Ig loci in mouse embryonic stem cells (EScells), such that chimaeric IgH and IgK loci were provided in whichhuman gene segments are functionally inserted upstream of endogenousconstant regions. To test if the human IgH-VDJ or IgK-VJ gene segmentsin the chimaera mice derived from human BAC-inserted ES cell clonesappropriately rearrange and express, RT-PCR was performed for the RNAsamples of white blood cells from those mice with the primer pairs ofhuman variable (V) region and mouse constant (C) region. The sequencesof oligos are shown as follows (Table 1). Each V oligo is paired with Coligo (HV with Cμ; KV with Cκ) for PCR reaction.

TABLE 1  Oligo Sequence HV2-5 AGATCACCTTGAAGGAGTCTGGTCC (SEQ ID NO 7)HV4-4 TGGTGAAGCCTTCGGAGACCCTGTC (SEQ ID NO 8) HV1-3CACTAGCTATGCTATGCATTGGGTG (SEQ ID NO 9) HV1-2ATGGATCAACCCTAACAGTGGTGGC (SEQ ID NO 10) HV6-1GGAAGGACATACTACAGGTCCAAGT (SEQ ID NO 11) CμTAGGTACTTGCCCCCTGTCCTCAGT (SEQ ID NO 12) KV1-9AGCCCAGTGTGTTCCGTACAGCCTG (SEQ ID NO 13) KV1-8ATCCTCATTCTCTGCATCTACAGGA (SEQ ID NO 14) KV1-6GGTAAGGATGGAGAACACTGGCAGT (SEQ ID NO 15) KV1-5TTAGTAGCTGGTTGGCCTGGTATCA (SEQ ID NO 16) CκCTTTGCTGTCCTGATCAGTCCAACT (SEQ ID NO 17)

Using the one-step formulation of SuperScript™ III One-Step RT-PCRSystem with Platinum® Taq High Fidelity (Invitrogen™; World Wide Web(www)invitrogen.com/site/us/en/home/References/protocols/nucleic-acid-amplification-and-expression-profiling/per-protocol/superscript-3-one-step-rt-per-system-with-platinum-taq-high-fidelity.html#prot3),both cDNA synthesis and PCR amplification were achieved in a single tubeusing gene-specific primers and target RNAs.

The RT-PCR results showed most of the human IGH-VDJ or IGK-VJ genesegments appropriately rearrange and express in the chimaera mice. Toinvestigate the details about the diversity generated from VDJ/VJrearrangement, those specific RT-PCR fragments were cloned into a commonvector for sequencing.

Sequencing results indicate that JH, DH, and JK usages (FIG. 35 and FIG.36) are similar to human results. In addition, the results from theIGH-VDJCμ transcripts show that the range and mean of CDR-H3 length(FIG. 37) are similar to that observed in human. The junctionaldiversity generated from exonuclease and nucleotide addition activities(FIG. 38) was also observed. The IGH rearrangement possessed a higherfrequency of these activities compared to the IGK one. These datasuggest that the inserted loci are functional in terms of generearrangement, junctional diversity as well as expression.

Example 6 Productive VJ Rearrangement and Somatic Hypermutation can beObtained

FIG. 41 shows an analysis of kappa mRNA from mice B-cells bearingrearranged VJ, the VJ having been rearranged from human germline kappaV1-8 and J1, and demonstrates that both that productive VJ rearrangementand somatic hypermutation can be obtained, the latter as seen from thechanges in antibodies encoded by mRNA with respect to the germlinesequences. The same is displayed for V1-6 and J1 in FIG. 42.Importantly, the recombination eliminates stop codons that are encodedby the combination of (unmutated) human germline gene segments, therebyallowing for antibody-encoding mRNA sequences. FIG. 43 demonstrates thatinserted human kappa V1-5 J1 and V1-5 J4 can produce functional codingsequences in vivo and junctional diversity.

Example 7 Inactivation of Use of Endogenous IGHV Gene Segments forExpressed Rearranged Heavy Chain by Inversion Introduction

A 5′-RACE Cμ-specific library was generated from the splenic Blymphocytes of transgenic mice, denoted S1 mice. These mice comprisetransgenic heavy chain loci, each locus containing the six most 3′functional human V_(H) gene segments (V_(H)2-5, 7-4-1, 4-4, 1-3, 1-2,6-1), and all the human D and J_(H) gene segments inserted into theendogenous heavy chain locus between endogenous IGHJ4 and Eμ (mousechromosome 12: between coordinates 114666435 and 114666436). The humanDNA was obtained from a bacterial artificial chromosome (BAC) containingthe sequence of human chromosome 14 from coordinate 106328951 tocoordinate 106494908. Further details on the construction of transgenicantibody loci using sRMCE is given elsewhere herein and in WO2011004192(which is incorporated herein by reference). 4×96-well plates of cloneswere randomly picked for sequencing to determine the usage of the genesegments. All detected immunoglobulin heavy chains were rearranged frommouse V_(H) or human V_(H) with human D-J_(H). No mouse D and J_(H)segments were detected in rearranged products (FIG. 44).

This result indicates that insertion of human V_(H)-D-J_(H) genesegments into an endogenous locus between the last endogenous J region(in this case, J_(H)4) and the Eμ enhancer effectively inactivates theuse of endogenous D and J_(H) gene segments for expressed rearrangedimmunoglobulin heavy chains.

The ratio of mouse V_(H) to human V_(H) usage was around 3 to 1 (FIG.45). To completely eliminate mouse V_(H) use for antibody generation,the endogenous mouse V_(H)-D-J_(H) was inverted and moved to a distantregion of the same chromosome. The rearrangement of mouse V_(H)S tohuman D-J_(H) segments was totally blocked by effects of inversion anddistance from the heavy chain locus.

The inversion strategy included three steps: (a) targeting of aninversion cassette, (b) inversion of endogenous VDJ and (c) excision ofmarkers (FIG. 46).

(a) Targeting of the Inversion Cassette:

The inversion cassette consists of four components: a CAGGSpromoter-driven puromycin-resistant-delta-thymidine kinase (puroΔtk)gene, a 5′ HPRT gene segment under the PGK promoter control, a loxP sitebetween them and inversely oriented to another loxP site already in theheavy chain locus, and two flanking piggyback LTRs (PB3′LTRs). Theinversion targeting cassette was inserted to a region that is 5′ anddistant to the endogenous IGH locus at chromosome 12 as shown in FIG.46. The targeted ES clones were identified and confirmed by PCR.

(b) Inversion:

Following the insertion, transient expression of cre from a transfectedplasmid resulted in inversion of a section of chromosome 12 fragmentincluding the endogenous V_(H)-D-J_(H) locus and intervening sequencesthrough recombination of two inverted loxP sites, ie, those in theinversion cassette and the landing pad for the BAC insertionrespectively. The invertants were selected by HAT and confirmed byjunction PCRs cross the two recombined loxP sites.

(c) Excision of Markers:

The inversion rearranged the relative orientation of the PB3′LTRs fromthe inversion cassette and PB5′LTR from the landing pad to generate twopiggyBac transposon structures flanking the inverted region. Withtransient expression of piggyBac transposase (PBase), these twotransposons were excised from the chromosome (and thus the mouse cellgenome). The cured ES clones were selected by1-(-2-deoxy-2-fluoro-1-b-D-arabinofuranosyl)-5-iodouracil (FIAU) and6TG, and confirmed by junction PCRs cross the excised regions.

Methods

Tissue Culture:

The procedures for ES cell culture, electroporation and drug selectionhave been described previously (Ramirez-Solis, R., A. C. Davis, and A.Bradley. 1993. Gene targeting in mouse embryonic stem cells. MethodsEnzymol. 225:855-878).

Targeting of the Locus for Inversion:

Briefly, 51 cell line (S1.11.1) was cultured in M15 medium (Knockout™DMEM supplemented with 15% fetal bovine serum, 2 mM glutamine,antibiotics, and 0.1 mM 2-mercaptoethonal). Targeting construct R57(FIG. 47) was linearized outside the region of homology by NotI. A totalof 20 μg of the linearized construct was electroporated into 51 celllines (AB2.1-derived) with a Bio-Rad® Gene Pulser™, and 107 cells wereplated onto three 90-mm-diameter SNL76/7 feeder plates containing M15medium. At 24 h after electroporation, M15 containing puromycin (3 μg ofthe active ingredient per ml) was added to each 90-mm-diameter plate,and the cells were maintained under selection for 9 days. 96puromycin-resistant clones were then picked and expanded in 96-wellplates. The targeting events were identified by long-range PCR.

Cre-loxP Mediated Inversion:

12 positive clones were pooled together and cultured in a 6-well tissueculture plate with M15 medium. The cells were transfected with 10 μg ofpCAGGS-Cre plasmid for the inversion of mouse endogenous locus and thenplated onto three 90-mm-diameter SNL76/7 feeder plates containing M15medium. At 24 h after electroporation, M15 containing 1XHAT(hypoxanthine-aminopterin-thymidine) was added to each 90-mm-diameterplate, and the cells were maintained under selection for 7 days and thentreated with 1×HT (hypoxanthine-thymidine) for 2 days. 48 HAT resistantcolonies were picked and genotyped by PCR amplification of the junctionsafter Cre-loxP mediated inversion.

HyPBase-Mediated Marker Excision:

12 positive clones were pooled together and cultured in E-well tissueculture plate using M15 medium. The cells were transfected with 5 μg ofHyPBase plasmid to activate the PB transposon LTRs flanking twoselection markers (Hprt-mini gene and PGK-puroΔtk gene) and plated ontoone 90-mm-diameter SNL76/7 feeder plates containing M15 medium. At 72 hafter electroporation, a serial dilution of the cells was then platedonto three 90-mm-diameter SNL76/7 feeder plates containing M15supplemented with1-(-2-deoxy-2-fluoro-1-b-D-arabinofuranosyl)-5-iodouracil (FIAU). Cellswere maintained under selection for 10 days, and FIAU-resistant colonieswere counted, picked, and expanded in 96-well plates. Positive cloneswere identified by PCR amplification of the junctions after excision ofthe selection markers. Positive clones were then expanded for blastocystmicroinjection.

Generation of Chimera and Breeding:

Mouse chimaeras were generated by microinjection of ES cells intoC57/BL6 blastocysts and transferred into pseudopregnant recipients. Malechimaeras were test-crossed with C57/BL6 mice. Agouti Fl offspring weregenotyped by 51 3′ junction PCR. Test-cross positive heterozygotes werefurther intercrossed to generate homozygotes.

Determination of VH-D-JH Usage by Rapid Amplification of 5′-cDNA Ends(5′ RACE) PCR:

Total RNA was extracted from the spleen of S1inv1 mouse (KMSF30.1d) withTRIzol® Reagent (Invitrogen™, Life Technologies Ltd™) and treated withDNase I. Rapid amplification of 5′-cDNA ends (5′ RACE) PCR was performedusing 573′ RACE kit (2nd Generation, Roche) following the protocolsupplied by the manufacturer. The first-strand cDNA was synthesisedusing primer E1554 (5′-ATGACTTCAGTGTTGTTCTGGTAG-3′; SEQ ID No 25) whichis located at the mouse endogenous Cμ region. The synthesised first cDNAstrand was purified using High Pure PCR Product Purification Kit(Roche). Poly(A) tail was added following the protocol supplied with the5′/3′ RACE kit (2nd Generation, Roche). The 5′ end of the V_(H)-D-J_(H)rearranged transcript was amplified by nested PCR with forward primersOligo dT, which is included in the kit, and nested Cμ-specific reverseprimers E1555 (5′-CACCAGATTCTTATCAGAC-3′; SEQ ID No 26). Followingreaction, the 5′ RACE PCR product was checked on a 1% agarose gel andpurified using QiAquick® Gel Extraction Kit (QIAGEN) as the protocolsupplied with the kit, then cloned into pDrive vector using QIAGEN PCRCloning Kit (QIAGEN) for sequencing analysis.

Results

The sequence analysis from a Cμ-specific 5′-RACE library of splenic Blymphocytes of S1^(inv1) (one human IGH BAC (ie, multiple human VH, allfunctional human D and JH) with an inverted endogenous IGH locusversion 1) mouse shows that practically all the transcripts came fromrearranged human V_(H)-D-J_(H) gene segments (FIG. 48). Mouse V_(H)usage was rarely detected (0.4%), and no mouse D and J_(H) usage wasdetected. Human V_(H) usage was 99.6% and only human D and J_(H) wereused; it was hypothesized that the rare mouse V_(H) usage was due totrans-switching with another chromosome and not due to use of moue V_(H)from the inverted sequences. The inversion resulted in completeinactivation of the endogenous V_(H) use.

This result indicates that inversion is an effective way to inactivatethe rearrangement of endogenous V_(H) gene segments. The S1^(inv1) mousealso shows a similar usage of both D and J_(H) gene segments to human(FIG. 49) (Link, J M et al. Mol. Immunol. 2005. 42, 943-955). Thus, amouse was produced that comprises a transgenic heavy chain locus thatexpresses heavy chains comprising human variable regions, but no mousevariable regions, and furthermore the human variable regionsdemonstrated a normal, human sequence distribution corresponding tohuman D and J usage observed in humans.

Example 8 Inactivation of Use of Endogenous IGHV Gene Segments forExpressed Rearranged Heavy Chain by Insertion of Human IgH Genomic DNAIntroduction

Insertion of human BACs with V_(H)-D-J_(H) gene segments into anendogenous mouse heavy chain locus between J_(H)4 and Eμ in chromosome12 allows human V_(H)-D-J_(H) gene segments to effectively use mouse Eμand 3′ enhancers and rearrange to generate chimeric antibody with humanvariable region and mouse constant region. Meanwhile, the endogenousV_(H)-D-J_(H) gene segments are pushed away from endogenous enhancersand constant regions. This distance effect results in inactivation ofmouse D and J_(H) use for expressed rearranged antibody products. As thedistance increases by stepwise BAC insertion, it is expected that themouse VH usage would be significantly reduced.

Results

Insertion of human DNA from a 1^(st) human BAC (BAC comprising a thesequence of mouse Chromosome 14 from coordinate 106328951 to coordinate106494908; containing six most 3′ functional V_(H) gene segments(V_(H)2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1), and all the human D and J_(H)gene segments) into the heavy chain endogenous locus of a AB2.1 ES cellgenome between endogenous IGHJ4 and Eμ (at mouse chromosome 12: betweencoordinates 114666435 and 114666436) effectively inactivates the use ofendogenous D and J_(H) gene segments for expressed rearrangedimmunoglobulin heavy chain (FIG. 44). The rearranged transcripts withmouse V_(H) gene segments are reduced in the resulting 51 mouse. Theproportion of transcripts using mouse V_(H) is around 75% of allobserved sequences (FIG. 45).

Following the 1^(st) BAC DNA insertion, human DNA from a 2^(nd) humanBAC (Chr14: 106494909-106601551) (BAC comprising a the sequence of mouseChromosome 14 from coordinate 106494909 to coordinate 106601551;containing 5 more functional VH gene segments (V_(H)3-13, 3-11, 3-9,1-8, 3-7)) was inserted into the landing pad left behind after curingfollowing the 1^(st) BAC insertion (see, eg, FIG. 24). The mouse V_(H)usage is further significantly reduced following this insertion of the2^(nd) BAC into the locus. The proportion of transcripts using mouse VHwas further reduced to 35% of all observed sequences (FIG. 50).

This result indicate that the endogenous V_(H)-D-J_(H) gene segmentscould be inactivated (ie, not used for expressed rearranged heavychains) through insertion of human VDJ sequences from one or more BACs.As the distance increases by stepwise BAC insertion, it is expected thatthe mouse VH usage would be significantly reduced.

Example 9 Normal Class Switch and Hypermutation in Transgenic Mice ofthe Invention Introduction

The B cell arm of the immune system has evolved to produce highaffinity, antigen-specific antibodies in response to antigenicchallenge. Antibodies are generated in B lymphocytes by a process ofgene rearrangement in which variable (V), diversity (D; for the IGHlocus) and joining (J) gene segments are recombined, transcribed andspliced to a Cμ (for IGH) or a Cκ or Cλ (for IGL) constant region genesegment to form an IgM antibody. Depending on the stage of B celldevelopment, IgM is either located on the cell surface or secreted. Therecombination process generates a primary antibody repertoire withsufficient germ line diversity to bind a wide range of antigens.However, it is usually not large enough to provide the high affinityantibodies that are required for an effective immune response to anantigen such as an infectious agent. Therefore, the immune system adoptsa two-stage diversification process to increase diversity further. Whenchallenged with antigens, B cells undergo selection and maturation by aprocess called somatic mutation. B cells expressing antibodies whichbind to antigen undergo multiple rounds of diversification, clonalexpansion and antigen selection in the germinal centres (GCs) of thesecondary lymphoid organs. During this process, the rearranged variableregions of the immunoglobulin genes acquire somatic hypermutationthrough nucleotide substitution, addition or deletion. This stepwiseprocess creates a secondary repertoire from the weak binders selectedoriginally from the primary repertoire and combines rapid proliferationof antigen-reactive B cells with intense selection for quality ofbinding, eventually giving rise to high affinity antibodies with broadepitope coverage. During this process, antibodies undergo classswitching in which the Cμconstant region is replaced by Cγ, Cα or Cε toproduce respectively IgG, A or E classes of antibody with differenteffector functions.

Insertion of 1^(st) human BAC (Chr14: 106328951-106494908) containingsix most 3′ functional V_(H) gene segments (V_(H)2-5, 7-4-1, 4-4, 1-3,1-2, 6-1), and all the D and J_(H) gene segments into the locus betweenendogenous IGHJ4 and Eμ (Chr12: 114666435 and 114666436) producestransgenic mice that generate chimeric immunoglobulin heavy chainscontaining human variable and mouse constant regions. This resultdemonstrates that human immunoglobulin gene segments are able to berearranged and expressed in mice. Here, RT-PCR experiments and sequenceanalysis were performed to further demonstrate that immunized transgenicmice have proper class switch and hypermutation for generatedantibodies.

Methods

RT-PCR and Sequence Analysis:

Wild type or S1 chimera mice at 6-8 weeks of age were primed byintraperitoneal injection of 10⁶ sheep RBCs suspended in phosphatebuffer saline (PBS). The immunized mice were boosted twice with the sameamount of sheep RBCs two and four weeks after priming. Four days afterthe last boost, peripheral blood cells were collected from the immunizedmice. Total RNA was isolated from peripheral blood cells with TRIzol®reagent (Invitrogen™) and treated with DNase I. Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using SuperScript® IIIFirst-Strand Synthesis System (Invitrogen™) following the protocolsupplied by the manufacturer. The 1st strand cDNA was synthesized withthe specific Cγ primers (Cγ1, Cγ2a, Cγ2b), following by PCR withspecific human V primers (VH1-2,3, VH4-4, VH6-1) and Cγ primers (Table2). Following reaction, the RT-PCR product was checked on a 1% agarosegel and purified using QiAquick® Gel Extraction Kit (QIAGEN) as theprotocol supplied with the kit, then cloned into pDrive vector usingQIAGEN PCR Cloning Kit (QIAGEN) for sequencing analysis.

TABLE 2  ELP1352_Cγ1 5′-AGAGCGGCCGCTGGGCAA SEQ ID CGTTGCAGGTGACGGTC-3′No 27 ELP1353_Cγ2b 5′-AGAGCGGCCGCTTTGTCC SEQ ID ACCGTGGTGCTGCTGG-3′No 28 ELP1354_Cγ2a 5′-AGAGCGGCCGCACATTGC SEQ ID AGGTGATGGACTGGC-3′ No 29ELP1356_VH4-4 5′-AGGACGCGTGAAACACCT SEQ ID GTGGTTCTTCCTCCTGC-3′ No 30ELP1357_VH1-2,3 5′-AGGACGCGTCACCATGGA SEQ ID CTGGACCTGGAGGAT-3′ No 31ELP1358_VH6-1 5′-AGGACGCGTATGTCTGTC SEQ ID TCCTTCCTCATCTTCC-3′ No 32

Results

The rearranged transcripts were detected using RT-PCR with humanVH-specific and mouse Cγ-specific primers for amplification fromperipheral blood cells of immunized transgenic mice (FIG. 51). Furthersequence analysis of these amplified fragments demonstratedhypermutation happened within the human variable regions of these IGγchains (FIG. 52). These results indicate that loci of the inventioncomprising insertion of human IGH BAC containing V_(H), D and J_(H) genesegments into the locus between endogenous IGHJ4 and Eμ regions hasnormal class switching and hypermutation functionality (IgM to IgG)following antigen challenge.

Example 10 Normal B Cell Compartments in Transgenic Mice of theInvention Introduction

In mice, about 2×10⁷ bone marrow immature B cells are produced daily.Among them, only 10-20% of these cells survive to exit the bone marrowand enter the spleen. The immature splenic B cell population is dividedinto two distinct subsets: transitional 1 (T1) and transitional 2 (T2) Bcells. In vivo experiments indicate that T1 cells give rise to T2 cells,whereas T2 cells can further differentiate into mature (M) B cells. Incontrast to immature B cells (3-4 days old), mature B cells arelong-lived (15-20 weeks old) and are ready to respond to antigens(Pillai S et al; Immunol. Reviews. 2004. 197: 206-218). Thus, thecomponent of mature B cell population is directly linked to theefficiency of humoral immune response.

The T1, T2 and M cell populations can be categorized by their cellsurface IgM and IgD levels. A normal phenotype of splenic B cellcompartment is required to mount a robust immune response.

Methods

Flow Cytometric Analysis of Mature B Lymphocytes:

To obtain a single cell suspension from spleen, the spleens of micelisted below were gently passaged through a 30 μm cell strainer. Singlecells were resuspended in PBS supplemented with 3% heat inactivatedfoetal calf serum (FCS; Gibco®). The following antibodies were used forstaining:

Antibody against B220/CD45R conjugated with allophycocyanin (APC)(eBioscience, clone RA3-6B2), antibody against IgD receptor conjugatedwith phycoerythrin (PE) (eBioscience, clone 11-26) and IgM receptorconjugated with fluorescein isothiocyanate (FITC) (eBioscience, clone11/41).

5×10⁶ cells were used for each staining. To each vial containingsplenocytes a cocktail of antibodies was added consisting of: IgD (PE)(eBioscience, clone 11-26), IgM (FITC) and B220/CD45R (APC). Cells wereincubated at 6° C. for 15 minutes, washed to remove excess of unboundantibodies and analysed using a fluorescence-activated cell sorting(FACS) analyser from Miltenyi Biotech. B-cells were gated asB220⁺IgM⁺IgD⁻ for T1 population, B220⁺IgM⁺IgD⁺ for T2 population andB220⁺IgM⁻IgD⁺ for M population. Percentage of cells was calculated usinggating system.

Results

Four different genotypes of mice were generated:

-   -   Wild type (WT);    -   A transgenic mouse homozygous for a heavy chain transgene        comprising insertion of the 1^(st) BAC human DNA noted above in        which there are 6 human VH, all functional human D and JH gene        segments (S1/S1);    -   A transgenic mouse homozygous for a heavy chain transgene        comprising insertion of a human VH, all functional human D and        JH gene segments (H1/H1); and    -   A transgenic mouse homozygous for a kappa chain transgene        comprising insertion of 6 functional human VK and 5 functional        JK gene segments (K1/K1).

Spleens from these naïve mice were collected and analysed for their Bcell compartments. The number and percentages of T1, T2 and M cellsamong those mice are similar (FIG. 53), indicating that geneticmanipulation of endogenous IG loci in transgenic mice according to theinvention do not compromise their B cell development. These data help toestablish that animals according to the invention provide a robustplatform for antibody discovery.

Example 11 Normal IgH Isotypes & Serum Levels in Transgenic Animals ofthe Invention

Transgenic mice (H1) carrying all human JH, all human DH and human Vh2-5under control of a rat switch region or mice (S1) carrying all human JH,all human DH and human Vh2-5, Vh7-41, Vh4-4, Vh1-3, Vh1-2 and Vh6-1under control of a mouse switch region were immunised with 100 μgCholera Toxin B subunit (CTB; Sigma-Aldrich® C9903) emulsified inComplete Freund's Adjuvant CFA; Sigma-Aldrich® F 5881). At least threeanimals were injected sc or ip and then boosted with 25 μg antigen inIncomplete Freund's Adjuvant (IFA; Sigma-Aldrich® F 5506) at (i) 14 daysand 21 days or (ii) 28 days after priming. Blood was taken beforepriming at day “−1” (pre-bleeds) and on the day the spleens were taken(usually 4d after last boost). Serum was analysed by ELISA using anantigen independent assessment of Ig isotypes. This assay detects totalserum antibodies of all species. Specific detection for mouse IgG1,IgG2a, IgG2b and IgM was used ((Anti-mouse IgG1 HRP AbD SerotecSTAR132P, Anti-mouse IgG2a HRP AbD Serotec STAR133P, Anti-mouse IgG2bHRP AbD Serotec STAR134P, Anti-mouse IgM HRP Abcam® ab97230) andconcentrations were read off a standard curve produced for each isotypeusing polyclonal isotype controls (IgG1, Kappa murine myelomaSigma-Aldrich® M9269, IgG2a, Kappa murine myeloma Sigma-Aldrich® M9144,IgG2b, Kappa from murine myeloma Sigma-Aldrich® M8894, IgM, Kappa frommurine myeloma Sigma-Aldrich® M3795). Results (FIGS. 54 & 55 for H1homozygous and S1 homozygous and heterozygous mice) showed that evenwith these relatively short immunisation regimes mice showed an increasein overall IgG levels after immunisation over pre-bleeds. In cases wherecontrol mice (+/+) not carrying any human immunoglobulin genes wereincluded and immunised, these mice showed comparable changes in totalobserved Ig levels (FIG. 54). Individual isotype levels were morevariable between animals possibly showing various stages of classswitching. IgM levels never exceeded 800 pg/ml whereas IgG levelsreached more than 6 mg/ml in some animals. Non-immunised controls showedno such increases in switched isotype Ig levels.

These results demonstrate that mice comprising multiple human VDJ genesegments under the control of a rat Sμ rat or mouse switch are able toundergo productive recombination and class switching in response toantigen challenge and that the mice produce antibody levels that arebroadly comparable to unmodified mice The transgenic mice are able toproduce antibodies of each of the IgG1, IgG2a, IgG2b and IgM isotypesafter immunisation. Titers for CTB-specific Ig in pre-bleeds andterminal bleeds were determined and all immunised animals showed atCTB-specific titres of at least 1/100 000.

Example 12 Generation of Anti-Ovalbumin Antibodies with Sub-50 nmAffinities from Animals of the Invention

Transgenic mice carrying all human JH, all human DH and human Vh2-5under control of a rat Sp switch region were immunised with 25 μgovalbumin (OVA; Sigma-Aldrich® A7641) in Sigma-Aldrich® adjuvant (SigmaAdjuvant System® S6322) ip and then boosted with the same amount of OVAin adjuvant at day 14 and day 21. Spleenocytes were taken 4 days laterand fused using 1 ml polyethyleneglycol (PEG Average MW1450;Sigma-Aldrich® P7306) with a myeloma line. Fused hybridoma cells wereplated on 5 96-well plates and after selection withhypoxanthine-aminopterin-thymidine (HAT) wells tested for expression ofOVA-specific antibodies by ELISA. Clones positive by ELISA werere-tested by surface plasmon resonance (SPR) and binding kineticsdetermined using the ProteOn™ XPR36 (Bio-Rad®). Briefly, anti-mouse IgG(GE Biacore™ BR-1008-38) was coupled to a GLM biosensor chip by primaryamine coupling, this was used to capture the antibodies to be testeddirectly from tissue culture supernatants. Ovalbumin was used as theanalyte and passed over the captured antibody surface at 1024 nM, 256nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone) used to doublereference the binding data. Regeneration of the anti-mouse IgG capturesurface was by 10 mM glycine pH1.7, this removed the captured antibodyand allowed the surface to be used for another interaction. The bindingdata was fitted to 1:1 model inherent to the ProteOn™ XPR36 analysissoftware. The run was carried out 1×HBS-EP (10 mM Hepes, 150 mM NaCl, 3mM EDTA, 0.05% polysorbate, pH7.6 (Teknova H8022)) used as runningbuffer and carried out at 25° C.

For 8 positive clones, heavy chain V-regions were recovered by RT-PCR(Access RT-PCR System, A1250, Promega) using forward primers specificfor Ig signal sequences (Wardemann et al Science 301, 1374 (2003)) andthe following reverse primers for the constant regions of mouse IgG(Table 3):

TABLE 3  Primer Name Sequence bp mlgG1_2 rev GGGGCCAGTGGATAGACAGAT 21SEQ ID No 33 mlgG2b rev CAGTGGATAGACTGATGG 18 SEQ ID  No 34 mlgG2a_2 revCAGTGGATAGACCGATGG 21 SEQ ID No 35 mCH1 unirev KCAGGGGCCAGTGGATAGAC 20SEQ ID No 36 mCH1 unirev_2 TARCCYTTGACMAGGCATCC 20 SEQ ID No 37

RT-PCR products were either directly sequenced using the same primerpairs or cloned in to TA plasmids (TOPO® TA Cloning® Kit for Sequencing,K4595-40, Invitrogen™) and submitted for plasmid sequencing. Results(Table 4, below) show that CDRH3 sequences had variable CDRs except fortwo identical clones (16C9 and 20B5) that also had near identical KDkinetic values. The determined equilibrium binding constant KD rangedfrom 0.38 nM to 40.60 nM, as determined by SPR at 25° C.

These results demonstrate that mice comprising multiple human VDJ genesegments under the control of a rat Cμ switch are able to undergoproductive recombination and produce high affinity antigen-specificantibodies whose CDR3 regions have sequences encoded by human genesegments (human JH was separately identified by V-Quest, IMGT).

TABLE 4  KD clone CDR3 and FR4 (underlined)  [nM] codeaccording to Kabat definition 0.38 16C9 QEVINYYYYGMDVWGQGTTVTVSS SEQ IDNo 38 0.52 20B5 QEVINYYYYGMDVWGQGTTVTVSS SEQ ID No 39 5.89 19F4LEMATINYYYYGMDVWGQGTMVTVSS SEQ ID No 40 39.70 19E1QEFGNYYYYGMDVWGQGTTVTVSS SEQ ID No 41 3.10 19G8 QEDGNPYYFGMDFWGQGTTVTVSSSEQ ID  No 42 8.95 20H10 GSSYYYDGMDVWGQGTTVTVSS SEQ ID  No 43 4.46 18D10LENDYGYYYYGMDVWGQGTTVTVSS SEQ ID No 44 40.60 16F2RGGLSPLYGMDVWGQGTTVTVSS SEQ ID No 45

Example 13 Generation of Anti-Cholera Toxin B Antibodies with Human VhRegions from Animals of the Invention

Transgenic mice carrying all human JH, all human DH and human Vh2-5,Vh7-41, Vh4-4, Vh1-3, Vh1-2 and Vh6-1under control of a mouse Sμ switchregion were immunised and fused as described in Example 11. Fusedhybridoma cells were plated on 5 96-well plates and after selection withhypoxanthine-aminopterin-thymidine (HAT) or G418 (Gibco® Cat No10131-027, Lot 503317) and wells tested for expression of CTB-specificantibodies by ELISA. Clones positive by ELISA were re-tested by surfaceplasmon resonance SPR and binding kinetics determined using the ProteOnXPR36™ (Bio-Rad®).

Briefly, anti-mouse IgG (GE Biacore™ BR-1008-38) was coupled to a GLMbiosensor chip by primary amine coupling, this was used to capture theantibodies to be tested directly from tissue culture supernatants.Cholera toxin B was used as analyte and passed over the capturedantibody surface at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM, with a 0 nM(i.e. buffer alone) used to double reference the binding data.Regeneration of the anti-mouse IgG capture surface was by 10 mM glycinepH1.7, this removed the captured antibody and allowed the surface to beused for another interaction. The binding data was fitted to 1:1 modelinherent to the ProteOn XPR36™ analysis software. The run was carriedout 1×HBS-EP (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate,pH7.6 (Teknova H8022)) used as running buffer and carried out at 37° C.

From the clones initially identified by ELISA, binding to CTB wasconfirmed by SPR. However, due to the pentameric nature of the choleratoxin B, the majority of fits to the 1:1 model were poor and theequilibrium binding constant KDs could not be accurately determined.Where fits were acceptable, equilibrium binding constant KDs determinedranged from 0.21 nM to 309 nM but due to the pentameric nature ofcholera toxin B these are likely to be the result of multimericinteractions and therefore apparent affinities with possible aviditycomponents.

Clones identified by SPR for binding to CTB were subjected to RT-PCR asdescribed in Example 12 to recover the Vh regions. RT-PCR products weredirectly sequenced using the same primer pairs. Results were obtainedfor only 14 clones presumably because the human primers described inWardemann et al were not designed to amplify mouse Vh regions andtherefore may have failed to amplify certain mouse Vh classes. Resultsshowed that 3 of the 14 CTB-specific recovered heavy chain V-regionsequences were human V, D and J regions as identified by V-Quest, IMGT(Table 5).

TABLE 5 Alignment of heavy chain CDRs and J-region of 3 clones identified as binding to CTB andpreferentially matching with human reference sequences from IMGT database; note that theKD values given here are apparent values due to the avidity of the CTB-antibody interactionVh Clone Sequence (Kabat definitions) KD region Name CDR1 CDR2 CDR3J-regions [NM] IGHV4- — SSNWWS EIYHSGSTNYNPSLKS n/a IGHJ2*01YWYFDLWGRGTLVTVSS — 4*02 (SEQ ID NO 51) (SEQ ID NO 56) (SEQ ID NO 64)12D10 SGNWWS EIYHSGNTNYNPSLKS GPLTGEKYYFDL -YYFDLWGRGTLVTVSS 0..27(SEQ ID NO 52) (SEQ ID NO 57) (SEQ ID NO 61) (SEQ ID NO 65) 1283 RSNWWSEIYHSGNTNYNPSLKS IGDWYFDL -WYFDLWGRGTLVTVSS 0.85 (SEQ ID NO 53)(SEQ ID NO 58) (SEQ ID NO 62) (SEQ ID NO 66) IGHV6- — SNSAAWNRTYYRSKWYNDYAVSVKS n/a IGHJ3*01 DAFDVWGQGTMVTVSS — 1*01 (SEQ ID NO 54)(SEQ ID NO 59) (SEQ ID NO 67) 4A12 SNSAAWN RTYYRSKWYNDYAVSVKSEGSHSGSWYLDAFDI DAFDIWGQGTKVTVSS 1.61 (SEQ ID NO 55) (SEQ ID NO 60)(SEQ ID NO 63) (SEQ ID NO 68)

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A host rodent whose genome comprises a chimeric immunoglobulin (Ig)locus, said chimeric Ig locus comprising human Tg locus DNA and host Iglocus DNA, said host Ig locus DNA comprising a host Ig upstream regionand a host Ig downstream region, said host Ig upstream region comprisinga host J region comprising a host J segment and said host Ig downstreamregion comprising a host Ig constant (C) region segment DNA comprising ahost C segment, wherein said human Ig locus DNA is positioned downstreamof said host Ig J region and upstream of said host Ig C region, saidhuman Ig locus DNA comprising one 10 or more human Ig variable (V)segments and one or more human Ig joining (J) segments.